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COLUMBIA UNIVERSITY DEPARTMENT OF PHYSIOLOGY THE JOHN G. CURTIS LIBR^
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WORKS OF J. A. MANDEL
PUBLISHED BY
JOHN WILEY & SONS.
A Tixt=book of Physiological Chemistry.
By Olof Hamtnarslen, Professor of Medical and Physiological Chemistry in the University of Upsala. Authorized translation, from the second Swedish edition and from the author's enlarged and revised German edition, by John A. Mandel, Assistant to the Chair of Chemistry, etc., in the Bellevue Hospital Medical College and in the Col- lege of the City of New York. 8vo, cloth, $4.00.
Handbook for Bio-Chemical Laboratory.
i2mo, cloth, $1.50.
rninnrjrrfKnn A TEXT-BOOK
OF
PHYSIOLOGICAL CHEMISTRY.
OLOF HAMMAKSTEN,
Professor of Medicul (ind Physiological Chemistry in the University of Upsala.
%x\ihoxxit^ Cninshition
FBOM THE AUTHOR'S ENLARGED AND REVISED FOURTH GERMAN EDITION
St
JOHN A. MANDEL,
Professor of Chemistry and Physics, and of Physiological Chemistry, in tfte New York University and Bellevue Hospital Medical College.
THIRD EDITION,
SECOND THOUSAND.
NEW YORK:
JOHN Wn.EY & SONS. London: CHAPMAN & HALL, Limited.
1901.
Copyright, 1900,
BY
JOHN A. MANDEL.
ROBERT DRUMMOND, PRINTER, NEW YORK.
PREFACE TO THE SECOND GERMAN EDITION.
After the appearance of the first Swedish edition of this text-book I "was asked by several colaborers abroad to provide a German translation, which was at that time impossible for several reasons. But I found it verv difficult to decline a similar proposal whicli I received from many col- leagues after the second edition appeared.
I yielded, therefore, to their expressed wishes; but I found after a time that it was impossible to obtain a translator in this special province of science, notwithstanding the unwearied exertions of my publisher. Nothing remained for me but to undertake the translation myself ; hence I ask the reader's indulgence for possible idiomatic or literal errors.
Specialists will at once perceive that the book before them is not a complete or detailed text-book. My intention was merely to supply students and physicians with a condensed and as far as possible objective representation of the principal results of physiologico-cheniical research and also with the principal features of physiologico-chemical methods of work. It seems to me that I have followed a common, practical, even if not strictly correct usage in allowing space in this book to the more important pathologico-chemical facts, although I have given the book the title Text- book of Physiological Chemistry.
The arrangement of subject-matter, which deviates considerably from that generally followed in text-books, was caused by the manner in whicli physiological chemistry is studied in Sweden. Here physiologico- and pathologico-chemical laboratory practice is obligatory on all students of medicine. In the arrangement of such practical work I continually kept in view that it should not consist of isolated, purely chemical or analytico- chemical problems, but that, as far as possible, it should always go hand in hand with tiie study of the different chapters of chemical physiology.
The study of physiologico-chemical processes within the animal body must precede the study of its component parts, its fluids and tissues; and this latter study, according to my experience, will then only inspire true
)ii
IV PREFACE TO THE SECOND GERMAN EDITION.
interest if. the study of the physiological significance of those component parts be closely pursued in connection with that of the transformations which take place in these fluids and tissues.
In view of this arrangement of subject-matter, and in order to render my book of greater interest and utility to those who do not wish to take cognizance of its analytico-chemical part, I have distinguished the latter by different setting of the type. With the exception of urinary analysis, which practically is of peculiar importance and which has been treated somewhat elaborately, this part in general depicts only the main points in the methods of- preparation and of analytical methods. The instructor who su23erintend& the laboratory practice and who chooses the problems for work has ample opportunity to give the beginner the necessary advanced directions, and for the more experienced student, as well as for the specialist, the excellent works of Hoppe-Seyler, Neubauer-Huppeet, and others render more explicit directions superfluous.
Olof Hammaesten". Upbala, October, 1890.
PREFACE TO THE THIRD GERMAN EDITION.
The present edition, which differs from tlie second in the arrangement of matter, contains three new chapters. The wonderful development of our knowledge of the chemistry of the carboliydrates in recent times has made it necessary to introduce a special chapter on this subject; and as the two chief groups of organic foods, the protein substances and the carbo- hydrates, are treated of in special chapters, the third group, the fats, like- wise has a chapter devoted to it. It also appears appropriate to treat the rather extensive subject of the chemistry of resjiiration in a special chapter and not, as heretofore, in connection with tlie blood. Another deviation from the earlier editions is that the present edition is supplied with the references to the literature, in pursuance of the request made on many sides. This edition is also thoroughly revised and enlarged according to tlie advance- ment of the science; still it was naturally impossible to incorporate into the text the various papers ai-)pearing or accessible to me during the printing of this edition.
Olof Hammarsten. Upsala, April, 1895.
V
PREFACE TO THE FOURTH GERMAN EDITION.
As this work is not a complete handbook, but only a concise text-book for students and physicians, I have considered it very desirable, in tlie preparation of this edition, not to enlarge the size of the volume. In view of the vast amount of new material supplied during the last four years, this task was a very difficult one, and its accomplishment was made j^ossible only by excluding those theories which in the light of recent researches have become obsolete, and by condensing some portions of the matter of the pre- vious edition. For this purpose a thorough revision of some of the chapters and a complete rewriting of others were necessary. By means of a new, space-saving arrangement of foot-notes the number of references to litera- ture has been increased. The original plan of the book, however, remains unchanged.
Olof Hammarsten.
Upsala, April 17, 1899.
TRANSLATOR'S PREFACE TO THE THIRD AMERICAN EDITION
Eecognizing the importance of keeping a text-book up to date, and especially one on a subject which is making such rapid advances as physi- ological chemistry, I was led to make a translation of the fourth German edition soon after the second American edition was issued. The aatlior's addenda have been incorporated into the text, bringing the available litera- ture up to April 1.
JoHX A. Mandel.
November, 1899.
Ti
CONTENTS.
CHAPTER I.
PAGE
Inthoduction 1
CHAPTER II. Protein Substances 15
CHAPTER III. Carbohydrates 71
CHAPTER IV. Animal Fats 92
CHAPTER V. The Animaj. Cell 99
CHAPTER VI. The Blood 123
CHAPTER VII. Chyle, Lymph, Transudations and Exudations 183
CHAPTER VIII. The Liver 206
CHAPTER IX. Digestion 249
CHAPTER X. Tissues of the Connective SuBST.JiNCE 316
CHAPTER XL Muscle 332
CHAPTER XII.
Brain and Nerves 358
vil
TLl CONTENTS.
CHAPTER XIII.
PAGE
Organs of Generation 370
CHAPTER XIV. Milk 385
CHAPTER XV. Urine 405
CHAPTER XVI. The Skin and its Secretions 521
CHAPTER XVII. Chemistry op Respiration , „.. . 530
CHAPTER ::VIII. Metabolism 546
PHYSIOLOGICAL CHEMISTRY.
CHAPTER I. INTRODUCTION.
It follows from the law of the conservation of force and matter that living beings, plants and animals, can produce neither new matter nor new force. They are only called upon to appropriate and assimilate already existing material and to transform it into new forms of force.
Out of a few relatively simple combinations, especially carbon dioxide and water, together with ammonium compounds or nitrates, and a few mineral substances, which serve as its food, the plant builds up the extremely complicated constituents of its organism, proteids, carbohydrates, fats, resins, organic acids, etc. The chemical work which is performed in the plant must therefore, in the majority of cases, consist in syntheses; but besides these, processes of reduction take place to a great extent. The kinetic energy of the sunlight induces the green parts of the plant to split off oxygen from the carbon dioxide and water, and this reduction is generally considered as the starting-point of the following syntheses. In the first place formaldehyde is produced, CO, + 11, 0 = CH,0 -f 0, , which then by condensation is transformed into dextrose, and this then serves in the structure of other bodies. The kinetic energy of the sun, which produces this splitting, is not lost; it is only transformed into another form of force — into the potential energy or chemical tension of the free oxygen on the one side, and the combinations less oxygenated, pioduced by the synthesis, on the other side.
Tliese conditions are not the same in animals. They are dependent either directly, as the herbivora, or indirectly, as the carnivora, upon plant- life, from which they derive the three chief groups of organic nutritive matter — proteids, carbohydrates, and fats. These bodies, of whicli the protein substances and fat form the chief mass of the animal body, undergo Avithin the animal organism a cleavage and oxidation, and yield as final
2 INTROD UCTION.
prodncts exactly the above-mentioned chief components of the nutrition of 2)lants, namely, carbon dioxide, water, and ammonia derivatives, which are ricli in oxygen and have little energy. The chemical tension, which is partly combined with the free oxygen and partly stored up in the above- mentioned more complex chemical compounds, is transformed into living force, heat, and mechanical work. While in the plant reduction processes and syntheses, which are active in the conversion of living force into potential energy or chemical tension, are the prevailing forces, we find in the animal body the reverse of this, namely, cleavage and oxidation ji recesses, which convert chemical tension into living force {vis viva).
This difference between animals and plants must not be overrated, nor must we consider that there exists a sharp boundary-line between the two. This is not the case. There are not only lower plants, free from chloro- phyll, which in regard to chemical processes represent intermediate steps between higher plants and animals, but the difference existing between the higher 2)lants and animals is more of a quantitative than a qualitative kind. Plants require oxygen as peremptorily as do animals. Like the animal, the plant also, in the dark and by means of those parts which are free from chlorophyll, takes up oxygen and eliminates carbon dioxide, while in the light the oxidation processes going on in the green parts are overshadowed or hidden beneath the more intense reduction processes. Like the animal the fermentive fangi transform chemical tension into living energy and heat; and even in a few of the higher plants — as the aroidece when bearing fruit — a considerable development of heat has been observed. The reverse is found in the animal organism, for, besides oxidation and splitting, reduc- tion processes and syntheses also take place. The contrast which seemingly exists between animals and plants consists merely in that in the animal organism the processes of oxidation and splitting are prevalent, while in the plant those of reduction and synthesis have thus far been observed.
AVoiiler' in 1824 furnished the first example of synthetical PROCESSES within the animal organism. He showed that when benzoic acid is introduced into the stomach it reappears as hippuric acid in the urine, after it combines with glycocoll (amido-acetic acid). Since the discovery of this synthesis, which may be expressed by the following equation,
C.H,.C001I + NH,.CH,.COOH = NH(C.H,.CO).CH,.COOH + H,0,
Benzoic acid Glycocoll Hippuric acid
and which is ordinarily considered as a type of an entire series of syntheses occurring in the body where water is eliminated, the number of known syntheses in the animal kingdom has increased considerably. Many of these syntheses have also been artificially produced outside of the organism,
' Berzelius, Lebrb. d. Cbemie, ilbersetzl von Wobler, Bd. 4, Dresden, 1831.
ANIMAL OXIDATIONS. S
and nnmerons examples of animal syntheses of which the course is abso- lutely clear will be found in the following pages. Besides tliese well-studied syntheses, there occur in the animal body also similar processes unquestion- ably of the greatest importance to animal life, but of which we know nothing with positiveness. We enumerate as examples of this kind of synthesis the reformation of the red-blood pigment (the haemoglobin), the formation of the different proteids from the peptones, the formation of fat from carbohydrates, and others.
Formerly tlie view was generally accepted that animal oxidatiox took place in the fluids, while to-day we are of the opinion, derived from the investigations of Pfluger and his pupils,' that it is connected with the form-elements and the tissues. The question how this oxidation in the form-elements proceeds and how it is induced cannot be answered with, certainty.
When a body is oxidized by neutral oxygen at ordinary temperature or at the temperature of the body, the body is called easily oxidized or auto- oxidized and the process is called a direct oxidation or autooxidation. As the oxygen of the inhaled air, as also of the blood, is neutral, molecular oxygen, the old assumption that ozone occurs in the organism has now been discarded for several reasons. On the other hand the chief groups of organic nutritives, carbohydrates, fat, and proteids, the last two forming the chief mass of the animal body, are not autooxidizable substances. They are on the contrary bradoxidizable (Traube) or dysoxidizable bodies. They are nearly indifferent to neutral oxygen, and it is therefore a question how an oxidation of these and other dysoxidizable bodies is possible in the. animal body.
In explanation it is very generally admitted that the oxygen is made active and this causes a secondary oxidation. It is generally conceded that in autooxidation a cleavage of neutral oxygen takes place. The autooxidiz- able substance splits the oxygen molecule and combines with one of the^ oxygen atoms, while the other free atom as active oxygen may oxidize the. simultaneously present dysoxidizable substances. Such a subordinate oxi- dation is called an indirect or secondary oxidation. The explanation of animal oxidations has been attempted by the supposition that the oxygen is made active and thus produces secondary oxidation.
The cause of the animal oxidation is considered, by PflCger and several other investigators, to be dependent upon the special constitution of the protoplasmic proteids. This investigator calls the proteids outside of the organism, and also those which circulate in the blood and fluids, " non- living proteids" as compared to those which are converted by the activity
' PflQger, Pfluger's Archiv. Bdd. 6 and 10 ; Finkler, ilnd., Bdd. 10 and 14 ; Oertman., ibid., Bdd. 14 and 15; Hoppe-Seyler, i/nd., Bd. 7.
4 INTRODUCTION.
of the living cell into living protoplasm, which he calls " living proteids'* or a special form of proteid called " active proteid " by Loew. It is now iilso considered that this "living proteid" differs from the "non-living proteid" by a greater mobility of the atoms within the molecule, and it may be characterized by a greater inclination towards intramolecular changes of position of these atoms. The reason for these greater intra- molecular movements Pfluger ascribes to the presence of cyanogen, LoEW to the presence of aldehydic groups, and Latham ' attributes it to the presence of a chain of cyanalcohols in the proteid mloecule.
Pfluger considers these differences between ordinary proteids and living protoplasmic proteids as the cause for the oxidation processes in the animal organism. These processes show certain similarity to the oxidation of phosphorus in an atmosphere containing oxygen. In this process the phosphorus is not only itself oxidized, but, as it splits the oxygen molecules and sets free oxygen atoms (active oxygen), it may cause at the same time an indirect or secondary oxidizing action upon other bodies present. In an analogous way the living protoplasmic proteid, which is not, like dead protpid, indifferent to molecular oxygen, may cause a splitting of the oxygen molecule, thus becoming itself oxidized, and at the same time setting oxygen atoms free, which may cause a secondary oxidation of other less oxidizable substances.
According to Pfluger the oxygen may be made active in this way. Active oxygen may also be produced, according to 0. Nasse, by a hydroxy- lization of the constituents of the protoplasm with the splitting off of mole- cules of water. If benzaldehyde is shaken with water and air an oxidation of the benzaldehyde into benzoic acid takes place, while oxidizable substances present at the same time may also be oxidized. The simul- taneous presence of potassium iodide and starch or tincture of guaiacnm causes a blue coloration because the hydroxyl (OH) takes the place of the hydrogen in the aldehyde group, and these two hydrogen atoms, one derived from the aldehyde and the other from the splitting of the water, have a splitting action on the molecular oxygen. Nasse and Eosing ' have found that certain varieties of proteid have the property of being hydroxylized in the presence of water, and they include among these proteids the substance philothion prepared by De Key-Pailhade ' from yeast and animal tissues
' PHDger's Arcbiv, Bd. 10 ; Loew and Bokorny, Pflliger's Arcbiv, Bd. 25 ; and Loew, ilnd., Bd. 30; O. Loew, The Energy of Living Protoplasm. London, 1896 ; — Latham, Britisli Medical Journal, 1886.
« O. Na«sc, Rostocker Zeitung, No. .'534, 1891, and No. 363, 189") ;— E. Rosing, Unter- stichungen liber die Oxydalion von Eiweiss in Gegenwart von Schwefel. Inaug. Dis. sert. Rostock, 1891.
^ De Rey-Pailhade. Recberches exper. surlePhilotbion, etc. Paris, 1891 ; — Nouvelles recherches sur Ic Pbilotbion. Paris, 1892 ;— and Chem. Centralhl., 1897, Bd, 2, S. 595.
ANnTAL OXIDATIONS. 6
and considered by him as an oxidation ferment. According to Nasse a whole series of oxidations in the animal body may be accounted for by the oxygen atoms set free in the hydroxylization similar to that of benzalde- hyde.
Another verywidely difTnsed view exists in regard to the origin of the activity of the oxygen, namely, that by the decomposition processes in the tissnes reducing substances are formed which split the oxygen molecule, uniting with one oxygen atom and setting the other free.
The formation of reducing substances during fermentation and putre- faction is generally known. The butyric fermentation of dextrose in which hydrogen is set free — C,H,,0, = C^II^O, -[- 2C0, + 2 (II J — is an example of this kind. Another example is the appearance of nitrates in consequence of an oxidation of nitrogen in cases of putrefaction, which process is ordi- narily explained by the statement that, in putrefaction, reducing, easily oxidizable bodies are formed which split oxygen molecules, liberating oxygen atoms which afterward oxidize the nitrogen. It is assumed, also that the cells of the animal tissues and organs have the property like these lower organisms, which cause fermentation and putrefaction, of causing splitting processes in which easily oxidizable substances, perhaps also hydrogen in statu nascendi (Hoppe-Seyler), are produced. The observa- tions of Ehrlich, that certain blue coloring matters — alizarin blue and indophenol blue — are decolorized by the tissues of the living animal and become blue again on exposure to air, seem also to be a proof of the occur- rence of easily oxidizable combinations in the tissues. A further proof of this is found in the observations of C. Ludwig and Alex. Schmidt,' that in the blood of asphyxiated animals, as well as in the absence of oxygen, an accumulation of reducing, easily oxidizable substances takes place.
In accordance with what has been stated above, we may assume that the oxidation in the animal body takes place in the following manner: The forces peculiar to protoplasm, unknown to us, but acting similarly to heat or the enzymes, cause a cleavage, producing reducing and readily oxidizable products on one side and difficultly oxidizable products on the other. The first may be directly oxidized, causing also a secondary oxidation of dysoxi- dizable bodies. The products formed by these splittings and oxidations may perhaps in part be burned within the body without undergoing further cleavage, but they must probably first undergo a further cleavage and then succumb to consecutive oxidation, until after repeated cleavage and oxida- tion the final products of metabolism are formed.
Nevertheless there are several investigators who do not admit of the snp-
' Hoppe-Seyler, Pflllger's Arcbiv, Bd. 13 ; P. Ehrlich, Das Sauerstoffbediivfniss des Organismus. Berlin, 1885 ; — Alex. Schmidt, Arbeiten aus der physiol. Anstalt zu Leipzig. 1867.
6 INTRODUCTION.
position of the oxygen becoming active. According to Traube, in antooxi- dation we have to deal in the first place, not with a cleavage of the oxygen, "but with a splitting of water in which the hydroxyl groups of the water combine with the oxidizable substance, while the hydrogen atom set free on the decomposition of the water unites with the neutral oxygen, forming hydrogen peroxide, which may naturally have an oxidizing action. Accord- ing to the view of Bach, which coincides essentially with the views of Eng- LER and Wild, oxygen atoms are not taken up in autooxidation, but entire oxygen molecules, which by the rupture of the double bonds of the oxygen
E— 0 yO
molecule form peroxide combinations with the formula, | or RY | .
E— 0 ^0
These can then, like hydrogen peroxide, give up an oxygen atom to a dy- ■soxidizable substance, passing into normal simple oxides R^O or E"0. Bach' ■explains in this way the oxidation process of the animal body.
Medyedew * has studied the conditions for the oxidation of salicylalde- hyde by tissue extracts. He has found on oxidation that two molecules of the above aldehyde react with oxygen instead of one. His investigations -also/coincide with the views of Bach, Engler, and Wild that a peroxide
C,H,.OH.C(/ -combination, ^ is produced as intermediate step in this
C.H,.OH.O^
^oxidation.
All the views presented thus far assume a continuous oxidation of the primary active substance. The view has also been suggested that animal oxidation may be brought about by oxygen-carriers, i.e., by bodies which, without being oxidized themselves, act in an analogous manner to the nitric oxide in the manufacture of sulphuric acid by alternately taking up and introducing oxygen in the oxidation of dysoxidizable bodies. Traube has for a long time explained the oxidations of the animal body in this way, and he calls these questionable oxygen-carriers oxidation ferments.'^
It has also been positively proven by the researches of Jaquet, Sal- KOwsKi, Spitzer, Eohmann, Abklous and Biarnes, Bertrand, Bou- QUELOT, De Eey-Pailiiade, Medvedew, Pohl,' and others, that in the
' M. Traube, Ber. d. deutsch. chem. Gesellsch., Bdd. 15, 18, 19, 22, and 26; Engler and Wild, ibid., Bd. 30 ; Bach, Le Mouiteur scientifique, 1897, and Compt. rend.. Tome 124.
' Plliiger's Arcliiv, Bd. 74.
^ M. Traube, Theorie dor Fermentwirkungeu. Berlin, 1858.
* Jaquet, Arch. f. exp. Path. u. Phurm., Bd. 29; Salkowski, Centralbl. f. d. med. ■Wissensch., 1892 and 1894 ; Virch()w'.s Arch., Bd. 147 ; Spitzer, Pfluger's Archiv, Bdd. >60 and 67; Spitzer and RObmann, Ber. d. deutsch. chem. Gesellsch., Bd. 28; Abelous
ANIMAL OXIDATIONS. 7
blood and different tissues of the animal body, as also in plant-cells, substiuices occur wliich have the property of causing certain oxidations and are therefore called oxidation ferments or oxidases. The exact knowledge of the nature of Jthese oxidation ferments has been somewhat advanced by Spitzer, who has been able to isolate ferruginous nucleoproteids from different animal organs, such as the liver, kidneys, testicles, pancreas, which act as oxygen-exciters. These proteids, whose iron Spitzer con- siders of special importance, readily decompose hydrogen peroxide, but they may also be detected in other ways, such as by tiie formation of indophenol from rr-naphthol and paraphenyldiamin in the presence of alkali. It is difficult at the present time to judge of the importance of the oxidation ferments wliich have been isolated from dead tissues, in the oxidation processes of the living animal body. Further investigations as to the nature and action of these bodies is very much to be desired.
LoEW,' who has opposed the view as to the oxygen becoming active with the setting free of oxygen atoms, has sought for the reason of the oxidations in the active proteid of the cells. The active movement of the atoms within the active proteid molecule is transmitted to the oxygen and to the oxidizable substance, and when the dissolution of the molecule has proceeded to a certain point the oxidation occurs by the chemical affinity. This oxidation is according to Loew a catalysis, which shows great analogy to the oxidation of alcohol under the influence of platinum-black.
Schmiedeberg,^ who also denies the supposition that the oxygen becomes active, is of the view that the tissue by the mediation of the oxida- tions do not increase the oxidizing activity of the oxygen, but more probably act on the oxidizing substances, making them more accessible to oxidation.
The many different views in regard to the oxidation processes show us strikingly how little positive is known about these processes. The occur- rence of numerous intermediary decomposition products in the animal body teaches us that the oxidations of the constituents of the body are not in- stantaneous and sudden, but take place step by step, and hand in hand with cleavages. Most investigators are agreed that these decompositions are similar to certain oxidations studied by Drechsel ' outside the animal body, where oxidations and reductions in quick succession acted together.
cl Biaru6s, Arch, de physiol. (5), Tomes 7, 8, and 9, and Compt. rend. see. bid.. Tome 46 ; Beitiaud, Arch, de physiol. (5), Tomes 8, 9, and Coinpt. rcud., Tomes 122, 133, 124 ; Bouiquelot, Compt. leud. soc. biol., Tome 48, and Compt. rend., Tome 123; De Rey- Pailhade. 1. c; Medvedew, Pflilger's Arch., Bd. 65; Pohl, Arch. f. exp. Path. u. Pharm., Bd. 38.
' O. Loew, The Energy of Living Protoplasm. Loudon, 1896.
» Arch. f. e.\p. Path. u. Pharm., Bd. 14.
•Jour. f. prakt. Chem. (N. F.), Bdd. 22, 29, 38, and C. Liidwig's Festschrift, 1887.
8 INTRODUCTION.
The views are divided in regard to the manner and origin of this coopera- tive action.'
The oxidations in the animal body have long been designated as a combustion, and snch a view is easily reconcilable with the above-mentioned views. In combustion in the ordinary sense, as, for example, the burning of wood or oil, we must not forget that the substances themselves do not combine with oxygen. It is only after the action of heat has decomposed these bodies to a certain degree that the oxidation of the products of such decomposition takes place and is accompanied by the phenomenon of light.
An important source of the living energy developed in the body is to be sought for in the oxidation effected by oxygen of strong potential energy, but CLEAVAGE PROCESSES are also important. In these complicated chemi- cal compounds are reduced to simpler ones, and therefore the atoms change from a labile equilibrium to a stabler one and stronger chemical affinities are satisfied, converting chemical potential energy into living energy {vis viva). The best-known example of such a splitting process outside of the animal organism is the ordinary alcoholic fermentation of dextrose, CjHj.O, = 2C0, + 2C,HgO, in which process heat is set free. The animal body^inay also have a source of energy in the cleavage processes which are not dependent on the presence of free oxygen. The processes taking ^^lace in the living muscle yield an example of this kind. A removed muscle, which gives no oxygen when in a vacuum, may, as Hermann ° has shown, work, at least for a time, in an atmosphere devoid of oxygen, and give off carbon dioxide at the same time.
We call cleavage processes which are accompanied by a decomposition of water and then a taking up of its constituents hydrolytic cleavages. These cleavages, which play an important role within the animal body, and which are most frequently met with in the processes of digestion, are, for example, the transformation of starch into sugar and the splitting of neutral fats into the corresponding fatty acid and glycerin :
C3H,(C,,H3.0J3 + 3H.0 = C,H,(OH), + 3(C,,H3eOJ.
Tristearin Glycerin Stearic acid
As a rule the hydrolytic cleavage processes as they occur in the animal body may be performed outside of it by means of higher temperatures with or without the simultaneous action of acids or alkalies. Considering the two above-mentioned examples, we know that starch is converted into sugar when it is boiled with dilute acids, and also that the fats are split into fatty acids and glycerin on heating them with caustic alkalies or by the action of superheated steam. The heat or the chemical reagents which
' See M. Nencki, Arch, des sciences biol. de St. P6toisbonrg, Tome 1, p. 483. » TJntersuchungen liber den StofEwechsel der Muskeln. Berlin, 1867.
FERMENTS AND ENZYMES. 9
are nsed for the • performance of these reactions would cause immediate death if applied to the living system. Consequently the animal organism must have other means at its disposal which act similarly, bnt in such a manner that they may work without endangering the life or normal consti- tution of the tissues. Sucli means have been recognized in the so-called %i7iorganize(l ferme)its or enzymes.
Alcoholic fermentation, as well as other processes of fermentation and putrefaction, is dependent upon the presence of living organisms, ferment fungi and splitting fungi of different kinds. The ordinary view, according to the researches of Pasteur, is that these processes are to be considered as phases of life of these organisms. The name organized ferments ov ferments has been given to such micro-organisms of which ordinary yeast is an example. However, the same name has also been given to certain bodies or mixtures of bodies of unknown organic origin which are products of the chemical work within the cell, and which after they are removed from the cell still have their characteristic action. Such bodies, for example malt diastase, rennin, and the digestive ferments, are capable in the very smallest quantity of causing a decomposition or cleavage in very considerable quantities of other substances without entering into permanent chemical combination Avitli the decomposed body or with any of the cleavage or decomposition i)roducts. These formless or unorganized ferments are generally called enzymes, according to Kuhxe.
A ferment in a more restricted sense is therefore a living being, while an enzyme is a product of chemical processes in the cell, a product which has an individuality even without the cell, and which may be active when separated from the cell. The splitting of invert-sugar into carbon dioxide and alcoliol by fermentation is a fermentative process closely connected with the life of the yeast. The inversion of cane-sugar is, on the contrary, an enzymotic process caused by one of the bodies or mixture of bodies formed by the living ferment, which can be severed from this ferment, and still remains active even after the death of the latter. Consequently ferments and enzymes are capable of manifesting a different behavior towards certain chemical reagents. Thus there exist a number of substances, among which we may mention arsenious acid, phenol, salicylic acid, boracic acid, sodium fluoride, chloroform, ether, and others, which in certain concentration kill ferments, but which do not noticeably impair the action of the enzymes.
The above view as to the difference between ferments and enzymes has lately been essentially shaken by the researches of E. Buciiner." He has been able to obtain from beer-yeast, by grinding and strong pressure, a cell fluid rich in proteid which Avhen introduced into a solution of a fermentable
' E. Buchner,' Ber. d. deutscb. chem. Qesellsch., Bdd. 30 and 31 ; E. Buchner aud Rapp. ibid., Bd 81.
10 INTRODUCTION.
sugar caased a violent fermentation. The objections' suggested from several sides that the fluid expressed still contained dissolved living cell substance has been answered by several important observations made by E. and H. Buchnee.' Among these observations we must mention the following: The active constituent of the cell fluid, zymase, is not influenced in its action by either chloroform or sodium arsenite solution (1^), while these bodies, on the contrary, completely destroy the fermentative action of the living yeast-cell. The activity of the zymase is not impaired by quantities of glycerin, which completely destroy fermentation produced by means of the yeast-cell. According to Buchi^er alcoholic fermentation is not directly connected with the organized structure of the cell, but pro- duced by soluble products secreted by the cells, or at least separated therefrom.
If the conclusions drawn by Buchneh from these important researches are correct, and if, as is to be expected, it can be applied to other micro- organisms, then we can understand the action of the above-mentioned anti- fermentative and anti-putrefactive substances in that they prevent the production of the active bodies by killing the cells or crippling their func- tions.y
As the enzymes may act outside of the cell, i.e., extracellular, still this does not preclude the possibility that we may also have enzymes which develop their action within the cell and are therefore intracellular. As an example of such an enzyme we may mention the enzyme existing in the micrococcus nrese, which has the power of decomposing urea, and also another enzyme, produced by a bacterium, which decomposes calcium formate into calcium carbonate, carbon dioxide, and hydrogen.
It is doubtful, indeed highly improbable, whether it has been possible up to the present time to isolate any enzyme in a pure state. Therefore the nature of the enzymes and their elementary composition are unknown. Such as have been obtained thus far appear to be nitrogenized and to be similar in some degree to proteid bodies. The enzymes are considered as proteid bodies by many investigators, but this opinion has not sufficient foundation. It is indeed true that the enzymes isolated by certain investi- gators act like genuine proteid bodies; but it is undecided Avhether or not the products isolated in these instances were pure enzymes or were com- posed of enzymes contaminated with proteids.
' II. Buchner, Silzungsber. d. Gesellscli. f. Morphol. u. Physiol, in Miincben, Bd. 13, 1897, Heft 1, which also contains the discussiim on this topic. See also Stavenlingtr., Ber. d. deutsch. Chem. Gesellsch., Bd. 30.
' The recent works on this disputed question may be found by referring to Abeles, Ber. d. deutsch. chem. Gesellsch., Bd. 31 ; Buchner and liapp, ibid., Bd. 33; Wro- blewski, Centralbl. f. Physiologie, Bd. 12. '
ENZYMES. 11
The enzymes may be extracted from the tissaes by means of water or glycerin, especially by the latter, which forms very stable solutions and consefjaently serves as a means of extracting them. The enzymes, generally speaking, do not appear to be diffusible. They arc readily carried down with other substances when these precipitate in a finely divided state, and this j^roperty is extensively taken advantage of in the preparation of pure enzymes.' The property of many enzymes of decomposing hydrogen peroxide is, according to Alex. Schmidt, not dependent upon the enzyme, but is caused by the contamination of the enzyme with constituents from the protoplasm. This coincides with tlie observations of Jacobsen " on emulsin, pancreas enzyme, and diastase, that the catalytic property may be destroyed by proper means without diminishing the specific euzymotic action. The continued heating of their solutions above + SO'"" C. generally destroys most of the enzymes. In the dry state, however, certain enzymes may be heated to 100'' or indeed to 150^-160°. C. without losing their power. The enzymes are precipitated from their solutions by alcohol.
We have no characteristic reactions for the enzymes in general, and each enzyme is characterized by its specific action and by the conditions under which it operates. But it must be stated that, however the different enzymes may vary in action, they all seem to have this in common, that by their presence an impulse is given to split more complicated combinations into simpler ones, whereby the atoms arrange themselves from an unstable equilibrium into a more stable one, chemical tension is transformed into living force, and new products are formed with lower heat of combustion than the original substance. The presence of water seems to be a necessary factor in the perfection of such decompositions, and the chemical process seems to consist in the taking up of tlie elements of water.
The action of the enzymes may be markedly influenced by external con- ditions. The reaction of the liquid is of special importance. Certain enzymes act only in acid, others, and the majority, on the contrary, act only in neutral or alkaline liquids. Certain of them act in very faintly acid as well as in neutral or alkaline solutions, but best at a specific reaction. The temperature exercises also a very important influence. In general the activity of enzymes increases to a certain limit with the temperature. This limit is not always the same, but depends, like the destructive action of high temperatures, essentially upon the quantity of enzyme and other con- ditions.' The products of the enzymotic processes exercise a retarding
' Brlicke, Wiener Sitziingsbericht, Btl. 43. 1861.
* AI. Schmiill, Zur Blutlebre. Leipzig, 1892 ;— Jucohsen, Zeitsclir. f. pbysiol. Chemie, Bd. 16, S. 340.
* Tammann, Zeitschr. f. pbysiol. Chem., Bd. 16, S. 271 ; Pugliesie, Pfliigers Arch., Bd. 69.
12 INTRODUCTION.
influence in proportion as they accamnlate. Additions of various kinds may have a retarding and others an accelerating action.'
An enzyme considered in the proper sense is one which has the property of producing hydrolytic cleavage. The three most important groups of these are the amylohjtic or diastatic, the p'^'oteolytic or those converting proteids into soluhle modifications, and the steatolytic or fat-splitting enzymes. Inverfin, which splits disaccharides into monosaccharides, belongs to the true enzymes, also the tirea- splitting and glucoside-sjjUtting enzymes, Avhich occur especially in higher plants. The proteid-coagtdating enzymes occupy a special position amongst the enzymes. The mode of action of these enzymes, amongst which we reckon chymosin (rennin), or casein-coagulating, and fibrin ferment, or blood-coagulating, is still less, known than the others. It is rather generally admitted that we here also have to deal with a hydrolytic cleavage, but still this has not been positively confirmed.
We are still in the dark in regard to the manner in which these enzymes act. Starting with the assumption that when the free ions are set free by the action of enzymes the electrical conductivity of the water must be raised, 0. XAsfeE" experimented with soluble starch, partly boiled and partly unboiled, and diastase, and determined the resistance according to Kohl- bausch's method and observed a considerable increase in the conductivity of the active diastase solutions. The enzymes by their action show in many regards a great similarity to so-called catalytic or contact action, and it is the generally accepted view that the enzyme action consists of a transfer of movement to the substance to be split.
As above stated, the enzymes are of great importance for the chemical processes going on in the digestive tract, but we have to add that the results of their action are greatly complicated by processes of putrefaction which take place in the intestine at the. same time, and which are caused by micro-organisms. Micro-organisms therefore exercise a certain influence on the physiological processes of the animal body. These organisms, when they enter the animal fluids and tissues and develop and increase, are of the greatest pathological importance, and modern bacteriology in relation to the doctrine of infectious diseases, founded by Pasteur and Koch, gives efficient testimony to these facts.
Putrefaction caused within the animal fluids and tissues by lower organisms may produce, among others, combinations of a basic nature. Such bodies were first found by Sei.mi in human cadavers, and called by him cadaver alkaloids or ptomaines. These ptomaines, which have been
' Fermi and Pernossi, Zeitsclir. f. TTygiene, Bfl. 18. An index of the literature on enzymes may be found v. in Moraczewski, PflUger's Arch., Bd. 69. » Rostocker Zttr., ?894.
PTOMAINES AND LEUCOMAINES. 13
isolated from cadavers aud some from putrefying proteid mixtures, have been closely studied by Selmi, Buiegeu, and Gautier ' and are cousideied as products of chemical processes caused by putrefaction microbes. The first ptomaine to be analyzed was coUidin, CJ[,,N, obtained by Nencki,' on the putrefaction of gelatin with pancreas. Since then many ptomaines have been analyzed by Gautier and especially by Brieger. Certain of the ptomaines originate undoubtedly from lecithin and other so-called extractives of the tissues, but the majority seem to be derived from the protein substances by decomposition.
Some ptomaines, although all belong to tlie aliphatic series, contain oxygen, and others are free from oxygen. The majority of the true ptomaines belong to the latter group. Most of the ptomaines isolated by Brieger are diamines or compounds derived from the same. Amongst the diamines we have two, cadaverin, or pentamethylendiamin, 0^11, ^N, , and jnitrescin, or tetramethylendiamiu, C^H,,Xj , which are of special interest because they have been found in the intestinal tract and urine in certain pathological conditions, namely, cholera and cystinuria.' Some of the ptomaines are exceedingly poisonous, while others are not. The poisonous ones are called toxines, according to the suggestion of Brieger.
The formation of such toxines in the decompositions caused by putrefac- tive microbes makes it probable that the lower organisms acting in infectious diseases also produce poisonous substances which may cause by their action the symptoms or complications of the disease. Brieger, who has become prominent by his study of this subject, has been able to isolate from typhoid cultures a substance called fyphotoxin, which has a poisonous action on animals; and he has also prepared another substance, teiafiin, from the amputated arm of a patient with tetanus, animals inoculated with which die exhibiting symptoms of developed tetanus.*
As above stated, the chemical processes in animals and plants do not stand in opposition to each other; they offer differences indeed, but still they are of the same kind from a qualitative standpoint. Pfluger says that there exists a blood-relationship between all living cells of the animal and vegetable kingdoms, and that they originate from the same root; and if the unicellular plant organisms can decompose protein sub-
' Selmi, Sulle ptomaine od alalcoidi cadaverici e loro importanza in tossicologia. Bologua, 1878. Ber. d. deutsch. cliem. Gesellscb., Bd. 11. Correspond, by H. Schiff ;— Brieger, Ueber Ptomaine, Parts 1, 2, and 3. Berlin, 1885-1886 ;— A. Gautier, Traite de chimie appliquce a la physiologic, Tome 2, 1873. Compt. rendus, Tome 94.
' Ueber die Zersetzung der Gelatine, etc. Bern, 1876.
* Brieger, Berlin, klin. "Wochenscbr., 1887; Baumann and Udransky, Zeitschr. f. physiol. Chem., Bdd. 13 and 15; Brieger and Stadthagen, Berlin, klin. Wochenscbr., 1889.
* Brieger, Virchow's Arch., Bdd. 112 and 115. Also Sitzuugsber. d. Berl. Akad. d. W., 1889, and Berl. klin. Wochenscbr., 1888.
14 INTRODUCTION.
stances in sach a manner as to produce poisonous substances, why should not the animal body, which is only a collection of cells, be able to produce under physiological conditions similar poisonous substances? It has been known for a long time that the animal body possesses this ability to a great extent, and as well-known evidence of this ability we may mention various nitrogenized extractives and poisonous constituents of the secretions of certain animals. Those substances of basic nature which are incessantly and regularly produced as products of the decomposition of the protein, substances in the living organism, and which therefore are to be considered as products of the physiological exchange of material, have been called Uucomaines by Gautier ' in contradistinction to the ptomaines and toxines produced by micro-organisms. These bodies, to which belong several well- known animal extractives, were isolated by Gautier from animal tissues such as the muscles. The hitherto known leucomaines, of which a few are poisonous in small amounts, belong to the cholin, the uric acid, and the creatinin group.
The leucomaines are considered as being of certain importance in caus- ing disease. It has been contended that when these bodies accumulate on account /of an incomplete excretion or oxidation in the system, an auto- intoxication may be produced (Bouchard' and others).
The toxines and the poisonous leucomaines are, however, neither the only nor the most active poison produced by the plant or animal cell. Later investigations have shown that certain plants as well as animals can produce proteids which are exceedingly poisonous. Such poisonous proteids have, for example, been isolated from the Jequirity and castor beans, as also from the venom of snakes, spiders, and other animals. The toxic proteids produced by pathogenic micro-organisms are of special interest. Bodies have been isolated from the cultures of various pathogenic microbes which are exceedingly poisonous and which reproduce the symptoms of infection more exactly than the toxines. These bodies, whose proteid nature is still questioned, have been called toxalbumins by Brieger and Frankel.
It is of great interest that we know also of proteid bodies such as the so-called alexines in the blood-serum, which have a germicidal or bacteri- cidal action. On the other hand we also have bodies of an alleged proteid nature which produce an immunity in the animal body against infection with a certain microbe or protection against the poison produced by the same microbe, so-called antitoxins. The great importance of these observa- tions is apparent, but as it is not within the range of this book we will not farther discuss the subject.
' Bull. soc. cbim., 43, and A. Gautier, Sur les alcaloYdes derives de la destruction hacterienne ou physiologique des tissus animaux. Paris, 1886.
« Bouchard, Le9ons sur les auto-iiitoxirations dans les maladies. Paris, 1887.
CHAPTER II. THE PROTEIN SUBSTANCES.
The chief mass of the organic constituents of animal tissues consists of amorphous, nitrogenized, very complex bodies of high molecular weight. These bodies, which are either proteids in a special sense or bodies nearly related thereto, take first rank among the organic constituents of the animal body on account of their great abundance. For this reason they are classed together in a special group which has received the name j^roteiu group (from TtpcoTevo, I am the first, or take the first place). The bodies belonging to these several groups are called profei7i sitbstafices, although in a few cases the proteid bodies in a special sense are designated by the same name.
The seveT'dl protein substances contain carbon, hydrogen, nitrogen, and oxygen. The majority contain also sulphur, a few phosphorus, and a few also iro7i. Copper, iodine, and bromine have been found in some few cases. On heating the protein substances they gradually decompose, producing inflammable gases, ammoniacal compounds, carbon dioxide, water, nitrogen- ized bases, as well as many other bodies, and at the same time they emit a strong odor of burnt horn or wool. On deep cleavage with acids they all yield, beside; nitrogenous bases, abundance of monoamido acids of different kinds.'
It is at present impossible to decide on a classification of the protein substances based upon their properties, reactions, and constitution, as well as upon their solubilities and precipitations, corresponding to the demands of science. The best classification is perhaps the following systematic summary of the better known and studied animal protein substances, due chiefly to Hoppe-Seyler and Drechsel."
' According to the view generally accepted up to the present time only those sub- stances are called true proteins which also yielded monoaraido acids on cleavage. The protamins will therefore bo discussed as an appendix to the protein substances.
* See "Eiweisskorper." Ladenbnrg's HandwOrterbuch derChemie. Bd. 3, S. 534-589, which gives a very complete summary of the literature of protein substances up to 1885.
15
16
TEE PROTEIN SUBSTANCES.
Albumins .
Globulins.
I. Simple Froteids or Albuminous Bodies.
( Seralbumin,
Ovalbumin,
Ladalbumin.
Fibrinogen^
Myosin,
Musculin, . Crystallin. j Casein,
\ Ovovitellin (^), and others, j Acid albuminate, \ Alkali albuminate. Albumoses (and Peptones).
j Fibrin,
\ Proteids coagulated by beat, and others.
Nucleo-albumins .
Albuminates
Coagulated Proteids .
Haemoglobins. Glycoproteids
Ifucleoproteids.
II. Compound Proteids.
Mucins and Mucinoids
Hyalogetis,
Amyloid,
Ichthulin, and others.
Helicoproteid. j Nucleohiston, \ Cytoglobin, and others.
III. Albumoids or Albuminoids.
Keratin.
Elastin.
Collagen.
Reticulin.
(Fibroin, Sericin, Cornein, Spongin, Conchiolin, BysBus, and others.')
To this summary must be added that we often find in the investigations of animal fluids and tissues protein substances which do not coincide with the above scheme, or do so only with difficulty. At the same time it must be remarked that bodies will be found which seem to rank between the different groups, hence it is very difficult to sharply divide these groups.
' Tlie classification of the proteins is a very difficult task, and no one has up to the present Mine been able to suggest such a classification free from exceptions. Under these circumstances, and as it appears desirable not to enlarge upon the existing uncertainty of the nomenclature in use, the author considers it unnecessary to change the aljove sum- mary. In regard to other classifications, see Ncumeister, Lchrbuch dor pliysiol. Chem., 2. Aufl., 1897, and Wroblewski, Ber. d. deutsch. chem. Gesellsch., Bd. 30.
SIMPLE P HOT EI D 8. 17
I, Simple Proteids c»r Albuiiiiiious Bodies.
The simple proteids are never-failing constituents of the animal and vegetable organisms. They are especially fonnd in the animal body, where they form the solid constituents of the muscles, glands, and the blood- serum, and they are so generally distributed that there are only a few animal secretions and excretions, sncli as the tears, perspiration, and perhaps urine, in which they are entirely absent or only occur as traces.
All albuminous bodies contain carbon^ hydrogen, nitrogen, oxygen, and sulphur ; ' a few contain a\so phosphorus. Iron is generally found in traces in their ash, and it seems to be a regular constituent of a certain group of the albuminous bodies, namely, the nucleo-albumins. The composition of the different albuminous bodies varies a little, but the variations are within relatively close limits. For the better studied animal proteids the following composition of the ash-free substance has been given:
C 50. G — 54. 5 per cent.
n G. 5 — 7.3
N 15.0 —17.6
S 0.3 — 2.2
P 0.42— 0.85 "
0 21.50 — 23.50 ''
A part of the nitrogen of the proteid molecule is easily split off as ammonia by the action of alkalies (Nasse). By the action of nitrous acid on protein substances only a very small part, 1-2 p.m., of the nitrogen is expelled, showing that only a small part thereof exists as amido groups in the protein molecule.' Hausmaxn ' has conducted investigations to show the distribution of the nitrogen in the proteid molecule. After boiling with hydrochloric acid he determined the amid nitrogen determinable as ammonia (a), then the nitrogen of the diamido bodies precipitable by phospho- tungstic acid {b), and the non-precipi table nitrogen of the monamido acids. He found the following percentages of the total nitrogen:
a b c
In crystallized ovalbumin ... . 8.53 21.33 67.80
" seralbumin 8.90 24.95 68.28
"casein 13.37 11.71 75.98
''gelatin 1.61 35.83 62.56
■ An exception is found in the mycoprotein of putrefaction bacteria and the anthrax- proteiu of the anthrax bacillus, which are sulphur-free proteids. See Nencki and Schaffer, Journ. f. prakt. Chem., Bd. 20 (N. F.), and Nencki, Ber. d. deutsch. chem. Gesellsch., Bd. 17.
" See Niisse. PtiQger's Arch., Bd. 6 ; Paal, Ber. d. deutsch. chem. Gesellsch., Bd. 29; Schiff, ibid., S. 1354, and O. Loew, Chemiker Zeit., 1896.
» Zeitschr. f. physiol. Chem., Bd. 27.
18 THE PROTEIN SUBSTANCES.
He found approximately 1-2^ amid nitrogen in true proteids, which is in accordance with the results of other investigators. A part of the sulphur separates as potassium or sodium sulphide on boiling with caustic potash or soda, and may be detected by lead acetate (Fleitmaxn, Danilewsky, Krugek, Fr. Schulz '). What remains can only be detected after fusing with nitre and sodium carbonate and testing for sulphates. The relationship between the sulphur split off by alkali to that not split off is different in various proteids. In most proteids thus far investigated the quantity of sulphur which can be split off amounts to a little less than one half of the total sulphur (Schulz). The profceid molecule therefore contains at least 2 atoms of sulphur. The molecular weight of the proteids is hard to determine accurately, and the results given for the same proteid, by various investigators, are often contradictory. The molecular weight is generally very high. For the alkali albuminate, in whose formation from native proteid a part of the nitrogen and sulphur is split off, LiEBERKUHX has given the formula 0,^11,^3^^3802,. In regard to the elementary formulse of proteids see Schmiedeberg.*
The constitution of the proteid bodies, notwithstanding numerous investigations, is still unknown. By heating proteids with barium hydrate and -v^^ter in sealed tubes at l50°-200° C. for several days, ScHUTZEisr- berger ^ obtained a number of products among which were ammonia, carbon dioxide, oxalic acid, acetic acid, and, as chief product, a mixture of amido-acids. This mixture contained, besides a little tyrosin and a few other bodies, chiefly acids of the series C^H^n+iNO^ (leucines) and CJI^n-iNO, {leuceines). The leucines and leuceines are formed from more complicated substances, with the general formula O^H^^^N^O^ , by hydrolytic splitting. These substances are called glucojjroteins by Schutzenberger on account of their sweet taste. The sulphur of the proteids yields sulphites. The three bodies, carbon dioxide, oxalic acid, and ammonia, are formed in the same relative proportion as in the decomposition of urea and oxamid; therefore Schutzbnberger suggests that perhaps proteid may be considered as a very complex ureid or oxamid. Such a conclusion cannot be derived from the above decomposition processes for several reasons.
On fusing proteids with caustic alkali, amn.onia, methyl-mercaptan, and other volatile products are generated; also leucin, from which then volatile fatty acids, such as acetic acid, valerianic acid, and also butyric
' Fleitmann, Annal. der Chem. und Pharm.. Bd. 66 ; Danilewsky, Zeitschr. f. pby- siol. Chem., Bd. 7 ; KrDgcr, PHiiger's Archiv, Bd. 43 ; F. Schulz, Zeitsclir. f, physioL Chem., Bd. 25. See also Suter, ibid., Bd. 20, and Drechsel, Centralbl. f. Physiol., Bd. 10, S. 529, in regard to forms of binding of the sulphur.
» Arch. f. cxp. Patli. u. Pharm., Bd. 39.
' Annal. do Cliim. el Phys. (5), 16, and Bull. soc. chira., 23 and 34.
CLEAVAGE PRODUCTS OF I'liOTEIDS. 19
acid, are formed; and tyrosin, from which hiter phenol, indol, and skatol are produced. On boiling with mineral acids (or still better by boiling with hydrocliloric acid and tin chloride, IIlasiwetz and TIahkkmaxn '), the proteids yield amido-acids, such as leucin, aspartic acid, glutamic acid, and tyrosin (and from vegetable albumin ScnuLzE and Barbieri " obtained «-phenylamidopropionic acid), also sulphuretted hydrogen, ethyl sulphide (Dkkchskl '), lencinimid,* ammonia, and nitrogenous bases (Dreciisel).
Amongst tlie bases obtained by Drechsel ' from casein, and by his pupils E. FisciiKK, ^I. Sik(JFriei), and S. IIedin from other proteids and gelatin on boiling with hydrochloric acid and tin chloride, we have one having the formula 0,11, ,N,0, or C,n,,N,0 + 11,0, which seems to be homologous to creatin or creatinin and called lysatin or lysatini7i by Drechsel. Another substance, called lijsin^ has the formula C,lI,^X,Oj. From its formula we find that it is homologous with ornithin, CjH,,X,0, (Jaffe), which it resembles in certain respects (see Appendix to this Chapter).
Besides these above-mentioned bases Hedin has obtained as cleavage products of different protein substances the bases arginin^ 0,11,^X^0, , first isolated by Schulze and Steiger from etiolated lupin and pumpkin seeds and also histidin, C.HgNjOj , prepared by Kossel from protamins^ Drechsel has also found diamido-acetic acid among the cleavage products of casein. On boiling with baryta-water both lysatinin and arginin yield urea among the other cleavage products, and it is therefore possible ta prepare urea from proteid by hydrolysis, without oxidation, making use of these bases as intermediary steps.
On the cleavage of the proteid, globin, contained in the hfemoglobin molecule, wMtli hydrochloric acid, Proscher' was able to regain about one half of the carbon, about one half of the nitrogen, two thirds of the hydrogen, and a little more than one half of the oxygen as tangible cleavage products. On the other hand R. Cohx ' has been successful in gaining about 97.8<i^ of the proteid (casein) as crystallizable or tangible cleavage jjroducts in his investigations on the quantitative proteid cleavage with hydrochloric
' Annal. d. Chem. u. Pharm., Bdd. 159 aud 169.
* Ber. d. deutscb. chem. Gesellsch., Ed. 16. » Central bl. f. Physiol.. Bd. 10.
* See Ritthausen, Ber d. deutscb. chem. Gesellsch., Bd. 29, and R. Cobu, Zeitschr. f. pbysiol. Chem., Bd. 22.
' Sitzuugsber. d. math.-pbys. Klasse d. k. saclis. Gesellsch. d. Wissenschaften, 1889. In the memoir " Der Abbau der Eiweissstoffe," Du Bois-Reymond's Arch., 1891, Drechsel gives a good review of his own investigations and of those of bis pupils. Fischer, Siegfried, aud Hedin. The literature of the above-meutioued bases will be given in the Appendix to this Chapter.
* Zeitschr. f. phj'siol. Chem., Bd. 27. ' Ibid., Bd. 26.
20 THE PROTEIN SUBSTANCES.
acid. He approximately calculated the leucin as -40-50,^ and the glutamic acid 30^. He obtained strikingly small quantities of basic products. He also found CO, and oxalic acid among the cleavage products of proteids with acid.
Proteids are decomposed by the action of proteolytic enzymes in the presence of water. First proteid bodies of lower molecular weight are formed — albumoses and peptones — and then on further decomposition amido-acids such as leucin, tyrosin, and aspartic acid. Both lysin, lysatinin, arginin, and histidin may be produced on far-reaching decomposi- tion (in tryptic digestion). On the extensive decomposition a chromogen may also be formed, which gives a violet color with chlorine- or bromine- water. This chromogen, which is formed in all far-reaching decompositions of proteids where leucin and tyrosin are formed, is called proteinocliromogen by Stadelmann, and trypto2}lian by Neumeister. Nencki ' considers this chromogen as the mother-substance of various animal pigments.
A great many substances are produced in the putrefaction of proteids. Pirst the same bodies as are formed in the decomposition by means of proteolytic enzymes are produced, and then a further decomposition occurs with the formation of a large number of bodies belonging to both the alipljatic and aromatic series. Belonging to the first series we have ammonium salts of volatile fatty acids, such as caproic, valerianic, and butyric acids, also succinic acid, carbon dioxide, methane, hydrogen, sulphuretted hydrogen, methyl-mercaptan, and others. The ptomaines also belong to these products and are probably formed by very different chemical processes or even syntheses.
E. Salkowski divides the putrefactive products of the aromatic series, into three groups: (a) the phenol group, to which tyrosin, the aromatic oxy -acids, phenol, and cresol belong; {l) the phenyl group, including phenylacetic acid and phenylpropionic acid; and lastly (c) the indol group, which includes indol, skatol, and skatolcarbonic acid. These various aromatic products are formed during the putrefaction with access of air. Nencki and Bovet' obtained only p.-oxyphenylpropionic acid, phenyl- propionic acid, and skatolacetic acid on the putrefaction of proteids by anaerobic schizomycetes in the absence of oxygen. These three acids are produced by the action of nascent hydrogen on the corresponding amido- acid, namely, tyrosin, jDhenylamidopropionic acid, and skatolamidoacetic acid, and these three last-mentioned amido-acids exist, according to Nencki, preformed in the proteid molecule.
' Stadelmann, Zeitscbr. f. Biologic, Bd. 26; Neumeister, ibid., Bd. 26, S. 329 ; Nciicki, Schweizer. Wocbenscbr. f. Pliarnmcie, 1891, and Ber. d. deutscli. chem. Ge- sellsch., Bd. 28.
* Salkowski, Zeit.schr. f. pbysiol. Cbeui., Bd. 12, S. 215 ; Nencki uiid Bovel, Monats- beft. f. Cbem.. Bd. 10.
DECOMPOSITION rno DUCTS OF I'liOTKIDS. 21
On distillation with snlphuric acid the 2)roteids yield a little fnrfurol, which indicates the presence of a carbohydrate group in the proteid mole- cule. According to Pavy even a carbohydrate, which he considers as animal gum, can be split oil from ovalbumin, and from this a reducing sub- stance is formed on boiling with an acid. This so-called carbohydrate is, according toWEYDEMAXX, certainly a nitrogenous substance, but Pavy has succeeded in obtaining the reducing substance directly from ovalbumin by- boiling with acid, and has prepared an osazon therefrom. This osazon, whose melting-point is 182°-185°, has been prepared by Krawkow ' from certain other proteids, and he therefore concludes that the carbohydrate group of the various proteids is the same. Tlie fact that a reducing carbo- hydrate can be split off from certain proteids, although small in amount, has been positively confirmed. Tiie splitting off of a carbohydrate is not possible from several pure proteids, such as casein, vitellin, myosin, and fibrinogen. Up to the present time it has been possible only when impure proteids, such as fibrin, or mixtures of various protein substances, such as lactalbumin, ovalbumin, or seralbumin were used. As example we may state that Spexzer, as well as K. Morxer, was unable to prepare a reducing carbohydrate from specially purified ovalbumin, while other investigators claim to have obtained said substance. This circumstance can perhaps be explained by the fact that the egg-albumin is a mixture of several sub- stances, among which is a glycoproteid, which has been prepared in a crystal- line state from ovalbumin by Hofmeister.'' The important question whether a carbohydrate group can be split off from pure proteids not contaminated with glycoproteids requires further proof.
EicHiioi.z ' has prepared an osazon from ovalbumin, which has a melting- point of 202°-20G°, while he was unable to prepare an osazon from either casein or seralbumin. Osazons have been prepared by Blumexthal and Meyer* from ovalbumin and also from the proteid of the yolk by boiling with acids. The osazon from the yolk had a melting-point of 203° and waa l»vo-rotatory, Avhile that from ovalbumin melted at 200°-205° and showed no positive hevo-rotatory power. These investigators do not consider the carbohydrate split off as an integral constituent of the proteid molecule. They rather consider the proteids yielding carbohydrates as glycoproteids, and this view is also accepted by Eichholz. J. See.maxn '" obtained 9f^
' Puvy, Tbe Pliysiolojry of the Carbohydrates. London, 1894 ; — Weydemaun, "Ueber den sog. thierisclie Ganimi," etc. Inaug.-Dissert. Marburg, 1896 ;— Kraw- kow, Pfliiger's Arch., Bd. 65.
'' SiKnizer, Zeitschr. f. phj'siol. Cheni., Bd. 24 : Morner, Centralbl. f. Phj'siol., Bd. 7; Hofmeister, Zeitschr. f. pbysiol. Chem., Bd. 24, S. 169.
' Journal of Physiol., Vol. 23.
* Ber. d. deutsch. chem. Gesellsch., Bd. 32.
* Bons' .\.rch. f. Verdaiiuntrskraiikheiteu. Bd. 4.
22 TEE PROTEIN SUBSTANCES.
redncing sabstance, calculated as dextrose, from ovalbumin. According to ^Juller's method he was able to prepare the h3^drochloric acid coni- iDination of this substance in question. From this behavior he draws the conclusion that carbohydrates split off by the action of acid are identical with the nitrogenous carbohydrate derivative glucosamine, obtained by him from ovomucoid, and by Muller from mucin.
On boiling with barium hydrate, or also in pepsin digestion, Fraxkel ' lias split off a nitrogenous substance from purified ovalbumin which gave neither a reaction witli Millox's reagent nor the Biuret reaction. It is readily soluble in water and dextro-rotatory. It does not directly reduce copper or bismuth salts, but does strongly reduce them on previously boiling with acid. The elementary analysis indicates the formula ^2(CgH30^.XII,) -p H 0, where n is generally represented by 2. Frai5"KEL considers it as a derivative of a biose and calls it "a/Zfamm" provisionally. He considers a chitosamin, which stands in close relationship to the osamin jirepared by Ml'LLER and Seemais:^' from mucin and ovomucoid, as the basis of this body.
In , marked contrast to all of these observations we have the communica- tion/of 0. Weiss.'' According to Pavt's alkali method he obtained a substance containing 1.8^ nitrogen, which yielded a reducing substance after boiling with acid. This reducing substance gave an osazon having a melting-point of 179°-191°. According to Weiss it is crystallizable methyl pentose with a melting-point of 91°-93° and isomeric with rhamnose.
B}^ the oxidaliou of proteids in acid solutions, volatile fattj"" acids, their aldehydes, iiitriles, ketones, as well as benzoic acid are obtained, also hydrocyanic acid by oxidizing with potassium dichromate and acid. Nitric acid gives various nitro-products, such as xanthoproteic acid (van dek Pants), triuitroalbumiu (Loew) or oxynilrualbumin, nilroben/.oic acid, and otliers. With aqua regia funiaric acid, oxalic acid, chlorazol, and other bodies are produced. By the action of bromine under strong pressure a large number of derivatives are obtained, such as bromanil and tribromacetic acid, bromo- forni, leucin. leuciiiimid, oxalic acid, tribromamido-benzoic acid, peptone, and bodies similar to humus.
Bytiiedry distillation of proleids we obtain a large number of decompo.sition products 'of a disagreeable burnt odor, and a porous glistening mass of carbon containing nitrogen ds left as a re-idue. The products of distillation are partly an alkaline liquid which con- tains ammonium carbonate and acetate, ammonium sulphide, ammonium cyanide, an infliimmable oil and other bodies, and a brown oil which contains hydrocarbons, nitro- gen ized bases belonging to the aniline and pyridine series, and a number of unknown substances.
It is impossible here to discuss all the products obtained by the action of different reagents on the proteids, but from the above-described decom- position products from proteids it is clear that the products belong in part to the fatty and in part to the aromatic series. Observers are not decided whether one or more aromatic groups exist preformed in the proteid mole- cule. According to Nencki the proteids contain three aromatic groups as
• Wien. Sitzungsber. Math.-naturw. Klasse, Bd. 107, Abth. II b.
* Central!)], f. Physiol., Bd. 12.
OXIDATION OF PliOTEIDS. 23
mentioned above: the tyrosin (oxyphenylamidopropionic acid), the phenyl- amidopropionic acid, and the skatolamidoacetic acid. Maly,' on account of the oxyprotosnlphonic acid prepared hy him, considers it not necessary to recognize more than one aromatic group in the jjroteid molecule.
By the oxidation of proteid by means of potassium permanganate, Maly obtained an acid, oxyprotosnlplionic acid, C 51.^1; H 0.89; N 14. .50; S 1.77; 0 "^5.54, which is not a cleavage product but an oxidation product in which the group HII is changed into SO,. Oil. This acid does not give the proper color reaction with Millox's reagent caused by aromatic hydroxyl derivatives (see below), nor does it yield the ordinary aromatic splitting products of the proteids. Still the aromatic group is not absent, but it seems to be in another binding from that in ordinary proteid. On oxidizing with potassium dichromate and acid this group a2)pears as benzoic acid, and on fusing with alkali benzol is given off.
On continuous oxidation a new amorphous acid, peroxyproteic acid — C 4G.:i2; H (i.4;3; N l->.:30; S 0.90; 0 34.09,1^— is produced from the oxy- protosnlphonic acid. The peroxyproteic acid gives the Buiret reaction, but is not precipitated by most of the reagents precipitating proteids.
According to Berxer'^ in the formation of oxyprotosulphonic acid not only does an oxidation take place, but also at tiie same time a deep cleavage due to the presence of alkali. He was able to show the presence of albu- moses and peptones as side products. These differed from the correspond- ing products produced in digestion by not yielding any indol or skatol on fusing with potash, by not giving ]\riLLOX's reaction, and not containing sulphur blackening lead. lie also found acetic acid, propionic acid, and butyric acid, and the presence of valerianic acid and basic bodies (lysin, histidiu) was shown among the cleavage products. On the cleavage of peroxyproteic acid with baryta he found the cleavage products previously obtained by Maly (with the exception of amidovalerianic acid and isogly- cerinic acid), besides also acetic, propionic, butyric acids, benzaldehyde and pyridin.
As in oxidation with potassium permanganate, so also may the proteids be changed by the action of the halogens, namely, so that they contain no sulphur which can be split off by alkali, or give Millox's reaction, nor yield tyrosin as a cleavage product. By the action of chlorine, bromine, and iodine on proteids the halogens pass into more or less firm union with the proteid (Loew, Blum, Blum and Vaubel, Liebreciit, Hop- kins and Brook, Hofmeister), and it is possible to prepare derivatives
' Sitzuiigsber. d. k. Akad. d. Wisseusch. Wieu, Abth. II, 1885, and Abth. II, 1888. Also MoniUshefte f. Cliem., Bdd. 6 and 9. See also Boudzynski and Zoja, Zeitacbr. f. pbysiol. Cheiu.. Bd. 19.
* Zeilschr. f. physiol. Cbem., Bd. 26,
24 THE PROTEIN SUBSTANCES.
with different bat constant quantities of halogen according to the method resorted to (Hopkins and Pinkus ').
On the putrefaction of proteids, as well as their decomposition by means of acids or alkalies and also by certain enzymes, among other products amido-acids are produced, and these have a certain significance for the probable formation of the proteids. It is more than likely that in the synthesis of proteids in the plant from the ammonia or the nitric acid of the soil, amido-acids or acid amids, among which asparagin plays an impor- tant role, are prodnced; and from these the albuminous bodies are derived by the action of glucose or other non-nitrogenized combinations.
The three basic bodies lysin, arginin, and histidin are formed, as shown by KossEL, as cleavage products of a gronp of bodies, the protamins, which were first shown by Miescher and then by Kossel to occur in fish- sperm as combinations of nucleic acid (see Chapter V). The protamins (see Appendix to this Chapter) are basic bodies which have some reactions in common with the proteids, but which yield no amido-acids on cleavage. As they yield the same basic products as proteids, they may, as suggested by KossEii, be considered to a certain extent as the nucleus of the proteid moledule, and the varions proteids may be derived from this nuclens by the addition of other atomic groups, monoamido acids and others.^
The question as to the preparation of proteid-like substances synthetically stands in close relation with the above stiitemeuts. In this connection we must mention in the first place the researches of Grimaux, and then SchDtzenberger and Pickerikq,' wha by the action of phosphorus penlchluride or peutoxiile on vaiious amido acids or by heat- ing aliiiie, were able to prepare bodies such as biuret, alloxan, xanthin, or ammonium substances either alone or mixed with other bodies. These substancos are similar in several ways with the proteids, although they cannot be considered as genuine pro- teids. The syntheses of gelatin or nlbumose-like substances published by Lilfen- FEivD'* will undoubtedly be of much greater importance when they have been substanti- ated by others.
The animal albuminous bodies are odorless, tasteless, and ordinarily amorphous. The crystalloid spherules {^Dotterpldttchen) occurring in the eggs of certain fishes and amphibians do not consist of pure proteids, but of proteids containing large amounts of lecithin, which seems to be combined
' Loew, Journ. f. prakt. Chem. (N. F.), Bd. 31 ; Blum, Mllnch. med. Wochenschr., 1896 ; Blum and Vaubel, Journ. f. prakt. Chem. (N. F.), Bd. 57 ; Liebrecht, Ber. d. deutsch. chem. Gesellsch., Bd. 30; Hopkins and Brook, Journ. of Physiol., Vol. 22; Hopkins and Pinkus, Ber. d. deiitsch. chem. Gesellsch., Bd. 31 : Hofmeister, Zeit.«rhr. f. physiol. Chem., Bd. 24.
' Kossel, Sitzungsber. d. Gesellsch. zur Beford. d. ges. Naturwissensch. zu Maibuig, No. 5, 1897, and Zeitschr. f. phy.siol. Chem., Bd. 25.
' See Pickering, Kings College, London, Physiol. Lab. Collect. Papers, 1897, where the works of Griina\ix are also cited ; also Journal of Physiol., Vol. 18, and Proceed. Roy. Soc. Vol. 60, 1897 ; Schiitzenberger, Compt. rend., Tomes 106 and 112.
* Du Bois-Peymond's Arch., 1894 ; Phy.siol. Abth.. S. 383 and 555.
REACTIONS OF TlIK PliOTEIDS. 25
with mineral substances. CrystuUine proteids ' liave been prepared from seeds of varions plants, and lately crystallized animal proteids (see seral- bumin and ovalbumin, Chapters \1 and XIII) have also been j)repared. In the dry condition the albuminous bodies appear as a white powder, or when in thin layers as yellowish, hard, transparent plates. A few are soluble in water, others only soluble in salt or faintly alkaline or acid solu- tions, while others are insoluble in these solvents. All albuminous bodies Avhen burnt leave an ash, and it is therefore questionable whether there exists any proteid body which is soluble in water without the aid of mineral substances. Xevertheless it has not been thus far successfully proved that a native albuminous body can be prepared perfectly free from mineral sub- stances without changing its constitution or its properties.' The albumi- nous bodies are in most cases strong colloids. They diffuse, if at all, only very slightly through animal membranes or parchment-paper, and the proteids therefore have a very high osmotic equivalent. All albuminous bodies are optically active and turn the ray of polarized light to the left.
On heating a proteid solution it is changed, the temperature necessary depending upon the proteid present, and with proper reactions of the solu- tion and nnder favorable external conditions — as, for example, in the presence of neutral salts — most proteids separate in the solid state as "coagulated" proteids. The different temperatures at which various proteids coagulate in neutral salt solutions give in many cases a good means of detecting and sejiarating these various bodies. The views in regard to the use of these means are divided.'
The general reactions for the proteids are very numerous, but only the most important will be given here. To facilitate the study of these they have been divided into the two following groups:
A. Precipitation Reactions of the Proteid Bodies.
1. Coagnlaiion Test. — An alkaline proteid solution does not coagulate ou boiling, a neutral solution only partly and incompletely, and the reaction
' See Maschke, Journ. f, prakt. Chem., Bd. 74; Drecbsel, ibid. (N. F.), Bd. 19; Griibler, ibid. (N. F.), Bd. 23 ; Ritthausen, ibid. (X. F.), Bd. 25 ; Scbmiedeberg, Zeit- scbr. f. pbysiol. Cbem., Bd. 1 ; Weyl, ibid., Bd. 1.
* See E. Haruack, Ber. d. deutscb. cbem. Gesellscb., Bdd. 23, 23, 25 ; Weiigo, Pflilger's Arcbiv, Bd. 48 ; Billow, ibid., Bd. 58.
* See Halliburton, Journ. of Pbysiol., Vols. 5 and 11 ; Coriu and Berard, Bull, de I'Acad. roy. de Belg., 15; Haycraft and Duggau, Brit. Med. Journ.. 1890, and Proc. Roy. Soc. Ed., 1889 ; Coria and Ansiau.x, Bull, de I'Acad. roy. de Belg., Tome 21 ; L. Fredericq, Centralbl f. Pbysiol., Bd. 3; Haycraft, ibid., Bd. 4 ; Hewlett, Journ. of Pbysiol., Vol. 13 ; Ducleu.K, Annal. Institut Pasteur, 7. In regard to the relationsbip of tbe neutral salts to tbe beat coagulation of albumins see also Starke, Sitzungsber. d. Gesellscb. f. Morpb. u. Pbysiol. in Miincben, 1897.
26 THE PROTEIN SUBSTANCES.
mnst therefore be acid for coagulation. The neutral liquid is first boiled and then the proper amount of acid added carefully. A lloccalent precipi- tate is formed, and if properly done the filtrate should be water-clear. If -dilute acetic acid be used for this test, the liquid must first be boiled and then 1, 2, or 3 drops of acid added to each 10-15 c. c, depending on the amount of profceid present, and boiled before the addition of each drop. If dilute nitric acid be used, then to 10-15 c. c. of the previously boiled liquid 15-20 drops of the acid must be added. If too little nitric acid be added, a soluble combination of the acid and proteid is formed which is precipitated by more acid. A proteid solution containing a small amount of salts must first be treated with about l<fo ISTaCl, since the heating test may fail, especially on using acetic acid, in the presence of only a slight amount of proteid. 2. Behavior toiuards Mineral Adds at Ordinary Temperatures. The proteids are precipitated by the three ordinary mineral acids and by metaphosphoric acid, but not by orthophosphoric acid. If nitric acid be placed in a test-tube and the proteid solution be allowed to flow gently thereon, a white opaque ring of precipitated proteid will form where the two liquids meet (Hellek's albumin test). 3. Precijjitation ly Metallic Salts: Copper sulphate, neutral and basic lead acetate (in small amounts), mercuric chloride, and other salts precipitate proteid. On this is based the •use of proteids as antidotes in poisoning by metallic salts. 4. Precipitation liy Ferro- or Ferncyanide of Potassium in Acetic Acid Solution. In these tests the relative quantities of reagent, proteid, or acid do not interfere with the delicacy of the test. 5. Precipitation by Neutral Salts, such as Na^SO^ or XaCl, when added to saturation to the liquid acidified with acetic acid or hydrochloric acid. 6. Precipitation ly Alcohol. The solution mast not be alkaline, but must be either neutral or faintly acid. It must, at the same time, contain a sufficient quantity of neutral salts. 7. Precipitation Tjy Tannic Acid in acetic-acid solutions. The absence of neutral salts or the presence of free mineral acids may not cause the precipitate to appear, but after the addition of a sufficient quantity of sodium acetate the precipi- tate will in both cases appear. 8. Precipitation hy Phospho-tungstic or PhospUo-molyhdic Acids in the presence of free mineral acids. Potassium,- mercuric iodide srnd potassiiwi-Msmiith iodide precipitate albumin solutions acidified with hydrochloric acid. 9. Precipitation by Picric Acid in solu- tions acidified by organic acids. 10. Precipitation by Trichloracetic Acid in 2-5^ solutions, and 11. by Salicylsulphonic Acid. The proteids are precipitated by nucleic acid, tanrocholic and chondroitin-sulphuric acid in acid solutions.
REACTIONS OF THE PROTEIDS. 27
B. Color Reactions for Proteid Bodies.
1. Millori^s reaction.^ A solntion of mercury in nitric acid containing some nitrous aciil gives a precipitate with proteid solutions which at the ordinary temperature is slowly, hut at the hoiling-point more quickly, colored red; and the solution may also he colored a feeble or bright red. Solid albuminous bodies, when treated by this reagent, give the same colora- tion. This reaction, whicli depends on the presence of the aromatic group in the proteid, is also given by tyrosin and other benzol derivatives with a hydroxyl group in the benzol nucleus.'' 2. Xatdhoproteic reaction. With strong nitric acid the albuminous bodies give, on heating to boiling, yellow Hakes or a yellow solution. After saturating with ammonia or alkalies the color becomes orange-yellow. 3. Adainkiewicz'' reaction. If a little proteid is added to a mixture of 1 vol. concentrated sulphuric acid and 2 vols, glacial acetic acid a reddish-violet color is obtained slowly at ordinary tem- l)eratnres, but more quickly on heating. Gelatin does not give this reaction. 4. Biuret test. If a proteid solution be first treated with caustic potash or soda and then a dilute copper sulphate solution be added drop by drop, first a reddish, then a reddish-violet, and lastly a violet-blue color is obtained. 5. Proteids are soluble on heating with concentrated hydrochloric acid, producing a violet color, and when they are previously boiled with alcohol and then washed with ether (Liebekmaxx ') they give a beautiful blue solution. 6. With concentrated sulphuric acid and sugar {in small quantities) the albuminous bodies give a beautiful red coloration. Eli,iott * has suggested the following as a reaction for protein substances. If dilute sulphuric acid (20 vols, in 100 vols, water) is allowed to act on the protein substances a bluish-violet color or a bluish-violet solntion is obtained on gradual concentration of the acid at ordinary temperature. Dilute hydrochloric acid acts in the same way. The solution shows a spectrum somewhat different from those obtained by Pettenkofer's, Liebermaxn's or Adamkiewicz's reactions. These color reactions apply to all albuminous bodies.
Mauy of these color reactions are obtained as shown by Salkowski ^ by the aromatic cleavage products of the proteids. Millon's reaction is only obtained by the substances of the phenol group ; the Xanthopkoteic reaction by the phenol group and skatol or
' The reagent is obtained in the following way : 1 pt. mercury is dissolved in 2 pts. of nitric acid (of sp. gr. 1.42), first when cold and later by warming. After complete solution of the mercury' add 1 volume of the solution to 3 volumes of water. Allow this to stand a few hours and decant the supernatant liquid.
* See O. Nasse, Sitzungsb. d. Naturforsch. Gesellsch. zu Halle, 1879 ; Vaubel and Blum, Journ. f. prakt. Chem. (N. F.). Bd. 57.
» Centralbl. f. d. med. Wissensch., 1887.
' .lourn. of Physiol.. Vol. 23.
' Zeitschr. f, physiol. Chem., Bd. 12, S. 215.
28 THE PROTEIN SUBSTANCES.
skatolcarbonic acid. Liebermakn's reaction is uot given by any of the :iromatic split- ting products. Adamkiewicz's reaction is only given by the iiidol group, especially skatolcarbonic acid. This reaction i., considered as a furfurol reaction bioughl about by a carbohydnue group as well as an aromatic group in the proteid. Liebekmann's reac- tion, as well as the reaction with sulphuric acid and sugar, seems at least to be a furfurol reaction. The biuret reaction is uot only given by proteid, pro'.amin and biuret, but also by artificially-prepared colloids (Gkimaux, Pickering) and many diauiids. Ac- cording to H. ScHiFF,' the presence of at least two groups (— CO.NHo) uiiiied in the molecule to a single atom of carbon or nitrogen, or by one or more groups (— C'O.NH) united in open chain. Both CO.NH3 groups mny also be directly united, as in oxaniid. Asparagin, a natural decomposition product of proteids, also gives the biuret reaction. Uobilin also gives a reaction similar to the biuret reaction, and the fact that a body gives the biuret reaction is not only sufficient proof of its being a protein.
The delicacy of the same reagent differs for the different albuminotis
bodies, and on this acconnt it is impossible to give the degree of delicacy
for each reaction for all albuminous bodies. Of the precipitation reactions
Heller's test (if we eliminate the peptones and certain albnmoses) is
recommended in the first place for its delicacy, though it is not the most
delicate reaction, and because it can be performed so easily. Among the
precipitation reactions, that with basic lead acetate (when carefully and
exactly executed) and the reactions 6, 7, 8, 9, and 11 are the most delicate.
The color reactions 1 to 4 show great delicacy in the order in which they are
given^
No proteid reaction is in itself characteristic, and, therefore, in testing for proteids one reaction is not sufficient, but a number of precipitation and color reactions must be employed.
For the quantitative estimation of coagulable proteids the determination by boiling with acetic acid can be performed with advantage, since, by operating carefully, it gives exact results. Treat the proteid solution with a 1-2^ common-salt solation, or if the solution contains large amounts of proteid dilute with the proper quantity of the above salt solution, and then carefully neutralize with acetic acid. Now determine the quantity of acetic acid necessary to completely precipitate the proteids in small measured portions of the neutralized liquid which have previously been heated on the water-bath, so that the filtrate does not respond with Heller's test. Now warm a larger weighed or measured quantity of the liquid on the water- bath, and add gradually the required quantity of acetic acid, with constant stirring, and continue tlie heat for some time. Filter, wash with water, extract with alcohol and then wnth ether, dry, weigh, incinerate and weigh again. With proper work the filtrate should not give Heller's test. This metliod serves in most cases, and especially so in cases where other bodies are to be quantitatively estimated in the filtrate.
The precipitation by means of alcohol may be used in the quantitative estimation of proteids. The liquid is first carefully neutralized, treated with some NaCl if necessary, and then alcohol added until the solution contains 70-80 vol. per cent anhydrous alcohol. The precipitate is collected on a filter after 24 hours, extracted with alcohol and ether, dried, weighed, incinerated and again Aveighed. This method is only applicable to liquids which do not contain any other substances, like glycogen, which are insolu- ble in alcohol.
' Ber. d. doutsch. chem. Gesellsch., Bd. 29.
SVNOrSLS OF PRO PERT IKS OF FROTEIDS. 2'J
In both these methods small fiuantities of proteids may remain in t!ie filtrates. These traces may be determined as follows: Concentrate the filtrate sulliciejitly, remove any separated fat by shaking with ether, and then precipitate with tannic acid. Approximately O;}^ of the tannic acid precipitate, washed with cold water and then dried, may be considered as proteid.
In many cases good resnlts may be obtained by precipitating all the proteid with tannic acid and determining the nitrogen in the washed pre- cipitate by means of K.teldaiil's method. On multiplying the quantity of nitrogen found by 0.25 we obtain the quantity of proteid.
The remov^al of proteids from a solution may in most cases be performed by boiling with acetic acid. Small amounts of proteid which remain in the filtrates may be separated by boiling with freshly precipitated lead carbonate or with ferric acetate, as described by IIofmeisteu.' If the liquid cannot be boiled, the proteid may be precipitated by the very careful addition of lead acetate, or by the addition of alcohol. If the liquid contains sub- stances which are precipitated by alcohol, such as glycogen, then tiie proteid may be removed by the alternate addition of potassium-mercuric iodide and hydrochloric acid (see Chapter VIII, on Glycogen Estimation), or also by trichloracetic acid as suggested by Ohermayer and Frankel.^
Synopsis of the Most Important Properties of the Different Chief Groups
of Proteids.
Those proteids which occur formed, in the ordinary sense, in the animal fiuids and tissues, and which can be isolated from these without losing their original properties by different chemical means, are called native proteids. Xew modifications, with other properties, may be obtained from these native proteids by the action of heat, various chemical reagents, such as acids, alkalies, alcohol, and others, as also by proteolytic enzymes. These new proteids are called modified ' proteids, in contradistinction to the native proteids. The albumins, globulins, and nucleoalbumins, as given in the scheme on page 1(3, belong to the native proteids, while the acid and alkali albuminates, albumoses, peptones, and the coagulated jiroteids belong to the modified proteids.
The native proteids may be precipitated by sufficient amounts of neutral salts without changing their properties, although the various proteids act differently with different neutral salts. Some are precipitated by NaCl, others only by MgSO^, and still others by only (NHJ^SO^, which is the precipitant for nearly all proteids. These various properties, as also the different solubility in water and dilute salt solution, are used at the present time to differentiate between the various proteids and groups, although it
' Zeitschr. f. physiol. Chem., Bdd. 2 and 4.
» Obermayer, Wieu. med. Jahrbiicher, 1888; Fritnkel, PflUger's Arch., Bdd. 52 and 55.
' The word denaturierung as used by Neumeister and the author is translated by the word modified, as it best expresses the meaning. The word derived might also be used.
30 TEE PROTEIN SUBSTANCES.
must be stated that these differences are only relative and are often uncertain.
Albumins. These bodies are soluble in water and are not precipitated by the addition of a little acid or alkali. They are precipitated by the addition of large quantities of mineral acids or metallic salts. Their solu- tion in water coagulates on boiling in the presence of neutral salts, but a weak saline solution does not. If NaCl or MgSO^ is added to saturation to a neutral solution in water at the normal temperature or at -f 30° C. no precipitate is formed; but if acetic acid is added to this saturated solution the albumin readily separates. When ammonium sulphate is added in substance to saturation to an albumin solution a complete precipitation occurs at ordinary temperature. Of all the albuminous bodies the albumins are the richest in sulphur, containing from 1.6^ to 2.2^.
Globulins. These albuminous bodies are insoluble in water, but dissolve in dilute neutral salt solutions. The globulins are precipitated unchanged from these solutions by sufficient dilution with water, and on heating they coagulate. The globulins dissolve in water on the addition of very little acid or alkali, and on neutralizing the solvent they precipitate again.
The/ solution in a minimum amount of alkali is precipitated by carbon dioxide, but the precipitate may be redissolved by an excess of the precipi- tant. The neutral solutions .of the globulins containing salts are partly or completely precipitated on saturation with NaCl or MgSO^ in substance at normal temperatures. The globulins are completely precipitated by saturat- ing with ammonium sulphate. The globulins contain an average amount of sulphur, not below Ifo.
A sharp line between the globulins on one side and the artificial albuminates on the other can hardly be drawn. The albuminates are, indeed, as a rule insoluble in dilute common-salt solutions ; but an albuminate may be prepared by the action of strong alkali which is soluble in common-salt solutions immediately after precipitatiou. We also have globulins which are insoluble in NaCl after having been in contact with water for some time.
Nucleoalbumins. This group of phosphorized proteids are found widely diffused in both the animal and vegetable kingdoms. The nucleoalbumins are found in organs abounding in cells, but they also occur in secretions and sometimes in other fluids in apparent solution as destroyed and altered protoplasm. The nucleoalbumins behave like rather strong acids; they are nearly insoluble in water, but dissolve easily with the aid of a little alkali. Such a solution, neutral or, indeed, a faintly acid one, does not coagulate on boiling. The nucleoalbumins resemble the globulins and the albumi- nates (see below) in solubility and precipitation properties, but differ from them in being hardly soluble in neutral salts. The most important differ- ence between the nucleoalbumins, the globulins, and the albuminates is that the nucleoalbumins contain phosphorus. They also differ from the other genuine proteids by this quantity of phosphorus and stand on this account
ALKALI AND ACID ALBUMINATES. 31
close to the nncleoproteids. They differ from the hitter in that thej do uot yield xunthiii bodies on cleavage. On peptic digestion most nucleo- albumins yield a proteid substance very rich in phosphorus, which has been called para- or pseudonuclein in contradistinction to the true nucleins (see Chapter \'). According to Likhermanx ' pseudonuclein is a combination of proteid with metaphosphoric acid. The nucleoalbumins seem to contain some iron.
The st'iKiratioii of pseudouuclein iu the peptic digestion of nucleoalbumins canuol be considered as positively chanicterislic of the nucleoalbuiniu group. The extent of such a cleavage is dependent upon the intensity of the pepsin digestion, upon the degree of acidity and tliu relationship between the nucleoalbuuiius and tiie digestive lluids. The separation of a pseudonuclein may, as shown by Salkowski, not occur even in the digestion of ordinary casein, and AVkoui.ewski did not obtain any pseudonuclein at all iu the digesti(.u of tlie casein from human milk. In the digestion of vegetable nucleo- alburnin Wiman'^ has also shown that the fact whether we obtain a great deal of pseudo- nuclein or not is dependent \ipon the way in which the digestion is performed. The most essential characteristic of this group of proteids is that they contain a given amount of phosphorus, and the absence of xantbin bases among their cleavage products.
The nucleoalbumins are often confounded with nncleoproteids and also with phosphorized glycoproteids. From the first class they differ by not yielding any xanthin bodies when boiled with acids, and from the second group by not yielding any reducing substance on the same treatment.
Lecithalbumins. In the preparation of certain proteih substances products are often obtained containing lecithin, and this lecithin can only be removed with difficult}' or incompletely by a mixture of alcohol and ether. Ovovitellin is such a protein body con- taining considerable lecithin, and Hoppe-Seyleh considers it a combination of proteid and lecithin. Liebermann^ has obtained proteids containing lecithin as an insoluble residue on the peptic digestion of mucous membranes of the stomach, liver, kidneys, lungs, and spleen. He considers them as combinations of proteid and lecithin and calls them lecithaLbuminn.
Alkali and Acid Albuminates. Xative proteids may, as the researches of recent date of several investigators such as Sjoqvist, 0. Cohxheim, BuGARSZKY and L. Lieberm-\.nx^ show, enter into combinations with acids and alkalies without changing their properties. On the contrary, by the sufficiently strong action of these reagents a modification may take place. By the action of alkalies all native albuminous bodies are converted, with the elimination of nitrogen or by the action of stronger alkali, also with the emission of sulphur, into a new modification, called alkali albuminate, whose specific rotation is increased at the same time. If caustic alkali in substance or in strong solution be allowed to act on a concentrated proteid solution, such as blood-serum or egg-albumin, the alkali albuminate may be
' Ber. (1. deutsch. chem. Gesellsch., Bd. 21.
' Salkowski, Ptillger's Arch., Bd. 63 ; — Wroblewski, Beitriige zur Kenntniss des Frauenkaseius. Inaug.-Diss. Bern, 1894; — Wiman, Upsala Liikaref. FOrh., N. F. 2.
" Hoppe-Seyler, Med. chem. Untersuch., 1868 ; also Zeitschr. f. physiol. Chem., Bd. 13, S. 479; Liebermann, Pflilger's Archiv, Bdd. 50 and 54.
* Sjoqvist, Skand. Arch. f. Physiol., Bd. 5 ; O. Cohuheim, Zeitschr. f. Biologie, Bd. 33 ; Bugarszky and Liebermann, Pfliiger's Arch., Bd. 72.
32 THE PROTEIN SUBSTANCES.
obtained as a solid jelly wliicli dissolves in water on heating, and which is called " Lieberkuhn's solid alkali albuminate." By the action of dilute caustic alkali solutions on dilute proteid solutions we have alkali albumi- nates formed slowly at the ordinary temperature, but more rapidly on heatin<7-. These solutions may be modified by the source of the proteid acted upon, and also by the extent of the action of the alkali, but still they have certain reactions in common.
If proteid is dissolved in an excess of concentrated hydrochloric acid, or if we digest a proteid solution acidified with 1-2 p. m. hydrochloric acid in the warmth, or digest the proteid alone with pepsin hydrochloric acid, we obtain new modifications of proteid which indeed may show somewhat vary- ing properties, bat have certain reactions in common. These modifications, which may be obtained in a solid gelatinous condition on sufficient concen- tration, are called acid albuminates or acid albumins, and sometimes syntonin, though we prefer to call that acid albuminate syntonin which is obtained by extracting muscles with hydrochloric acid of 1 p. m. F. GoLD- SCHMIDT ' has shown in the action of acids on ovalbumin that even in very
dilute solutions of acid f:T-;HCl) secondary albumoses are produced at the \1G /
same time as acid albuminates, which shows that the acid albuminate forma- tion ia accompanied by the splitting off of albumoses. He also found that the formation of secondary albumoses did not require the previous formation of primary albumoses. The extent as to the formation of acid albuminate, hemiprotein (Kuhne's antialbuminate), various albnmoses, peptones, and further cleavage products is essentially dependent upon the temperature and upon the concentration of the acid.
The alkali and acid albuminates have the following reactions in common: They are nearly insoluble in water and dilute common-salt solu- tion (see page 30), but they dissolve readily in water on the addition of a Tery small quantity of acid or alkali. Such a solution or one nearly neutral does not coagulate on boiling, but is precipitated at the normal temperature on neutralizing the solvent by an alkali or an acid. A solution of an alkali or acid albuminate in acid is easily precipitated on saturating with NaCl, but a solution in alkali is precipitated with difficulty or not at all, according to the amount of alkali it contains. Mineral acids in excess precipitate solutions of acid as well as alkali albuminates. The nearly neutral solutions of these bodies are also precipitated by metallic salts.
Notwithstanding this agreement in the reactions, the acid and alkali albuminates are essentially different, for by dissolving an alkali albuminate in some acid no acid albuminate solution is obtained, nor is an alkali
• Ueber die Einwirkung von Siluron auf Eiweissstoffe. Inaug.-Diss. Strassburg, 1898.
ALBUMOSES AND PEPTONES. 33
albuminate formed on dissolving an acid albuminate in water by the aid of a little alkali. In the first case we obtain a solution of the combination of the alkali albuminate and the acid and in the other case a soluble combina- tion of the acid albuminate with the alkali added. The chemical process in the modification of proteids with an acid is essentially different from the modification with an alkali, hence the products are of a different kind. The alkali albuminates are relatively strong acids. They may be dissolved in water with the addition of CaCO,, with the elimination of CO,, which does not occur with typical acid albuminates, and they show in opposition to the acid albuminates also other variations which stand in connection with their strongly marked acid nature. Dilute solutions of alkalies act more energetically on jjroteids than do acids of corresponding concentration. In the first case a part of the nitrogen, and often also the sulphur, is split off, and from this property we may obtain an alkali albuminate by tlie action of an alkali upon an acid albuminate; but we cannot obtain an acid albumi- nate by the reverse reaction (K. MoRNER '), For this reason the calling of the modified proteid obtained by the action of alkali or acid, protein, and the combinations of this protein with alkali, alkali albuminate and the combination with acid, acid albuminate, leads to a misunderstanding or to a wrong conception.
Desamidoalbuminic acid is au alkali-albuminate which Schmiedeberg' obtaiued by llie actiuu of such weak alkali that a pail of the uitrogen was evolved, but the quantity of sulphur remained the same. The proteid combination obtaiued by Blum bj' the action of formol on proteid and called by bim protogen,'^ has similarities with the alkali-albu- miuates in regard to solubilities and precipitation, but is not identical therewith.
The preparation of the albuminates has been given above. By the action of alkalies or acids upon a proteid solution the corresponding albuminate may be precipitated by neutralizing with acid or alkali. The washed precipitate is dissolved in water by the aid of a little alkali or acid, and again precipitated by neutralizing the solvent. If this precipitate which has been washed in water is treated with alcohol and ether, the albuminate will be obtained in a pure form.
Albumoses and Peptones. Peptones are designated as the final products of the decomposition of albuminous bodies by means of proteolytic enzymes, in so far as these final products are still true albuminous bodies, while we designate as albumoses, proteoses, or propeptones the intermediate products produced in the peptonization of proteids in so far as they are substances not similar to albuminates. Albumoses and peptones may also be produced by the hydrolytic decomposition of the proteids with acids or alkalies, also by the putrefaction of the same. They may also be formed in very small
' Pdilger's Archiv, Bd. 17.
• Arch. f. exp. Path. u. Pharm., Bd. 39.
' Blum, Zeitschr. f. physiol. Chera., Bd. 22. The older investigations of Loew may be found in Maly's Jahresber., 1888. On the action of formaldehyde, see also Benedi- centi, Du Bois-Rcvmond's Arch., 1897.
34 THE PROTEIN SUBSTANCES.
qnantities as by-prodncts in the investigations of animal fluids and tissues, and the question to what extent these exist preformed under physiological conditions requires very careful investigation.
Between the peptone which represents the final cleavage product and the albumose which stands closest to the original proteid we have undoubtedly a series of intermediate products. Under such circumstances it is a difficult problem to try to draw a sharp line between the peptone and the albumose group, and it is just as difficult to define our conception of peptones and albumoses in an exact and satisfactory manner.
The albumoses have been considered as those albuminous bodies whose neutral or faintly acid solutions do not coagulate on boiling and which, to distinguish them from peptones, were characterized chiefiy by the following properties. The watery solutions are precipitated at the ordinary tempera- ture by nitric acid as well as by acetic acid and potassium ferrocyanide, and this precipitate has the peculiarity of disappearing on heating and reappear- ing on cooling. If a solution of albumoses is saturated with NaCl in substance, the albumoses are partly precipitated in neutral solutions, but on the addition of acid saturated with the salt they completely precipitate. This precipitate, which dissolves on warming, is a combination of albnmose with the acid.
"We formerly designated as peptone those proteid bodies which are readily soluble in water and which do not coagulate by heat, whose solutions are precipitated neither by nitric acid, nor by acetic acid and potassium ferro- cyanide, nor by neutral salts and acid.
The reactions and properties which the albumoses and peptones had in common were formerly considered as the following: They give all the color reactions of the proteids, but with the biuret test they give a more beautiful red color than the ordinary proteids. They are precipitated by ammoniacal lead acetate, by mercuric chloride, tannic, phospho-tungstic, phospho- molybdic acids, potassium-mercuric iodide and hydrochloric acid, and lastly by picric acid. They are precipitated but not coagulated by alcohol, namely, the precipitate obtained is soluble in water even after being in contact with alcohol for a long time. The albumoses and peptones alsa have a greater diffusive power than native albuminous bodies, and the diffusive power is greater the nearer the questionable substance stands to the final product, the now so-called jiure peptone.
These old views have undergone an essential change in the last few years. After IIeynsius' ' observation that ammonium sulphate was a general precipitant for proteids, also peptone in the old sense, Kuiine" and
• P Auger's Archiv, Bd. 34.
2 See Kilbnc, Verliandl. d. naturhistor. Vereins zu Heidelberg (N. F.), 3 ; J. Wenz, Zeitschr. f. Biologie, Bd. 23 ; KUlme and Cbitteuden, Zeitschr. f. Biologie, Bd. 23; R. Neumeister, ibid., Bd. 23 ; Kiihue, ibid., Bd. 29.
ALBU MOSES AND PEPTONES. 35
his pupils proposed this salt as a means of separating albumoses and peptones. Those products of digestion which separate on saturating their solution with ammonium sulpliate are considered by KCuxe and indeed by most of the modern investigators as albumoses, while those which remain in solution are called peptones or pure peptone. This pure peptone is formed in relatively large amounts in pancreatic digestion, while in pepsin digestion it is only formed in small quantities or after prolonged digestion.
According to Schutzexbeugeu and Kuhxk' the proteids yield two chief groups of new albuminous bodifes Avhen decomposed by dilute mineral acids or with proteolytic enzymes; of these the anti group shows a greater resistance to further action of the acid and enzyme than the other, namely, the hcmi group. These two groups are, according to Kuiixe, united in the dilferent albumoses, even though in various relative amounts, and each albumose contains the anti as Avell as the hemi group. The same is true for the peptone obtained in pepsin digestion, hence he calls it amplioi^eptone. In tryptic digestion a cleavage of the amphopeptone takes place into anti- peptone and liemii)eptone. Of these two peptones the hemipeptone is further split into amido acids and other bodies while the antipeptone is not attacked. By the sufficiently energetic action of trypsin only one peptone is at last obtained, the so-called antipeptone. According to the researches of Kutscher' the antipeptone obtained in the pancreatic digestion is not a chemical individuality, but a mixture in which the hexon bases histidin and arginin, besides monamido acids, have been detected. This also follows from the observations made by Balke that the antipeptone prepared by him could be separated into two parts by phospho-tungstic acid, one part rich in bases and the other rich in acids. For these reasons Kutscher also denies the chemical individuality of carnic acid (see page 4o), which Siegfried and Balke consider as identical with antipeptone. "With this view the work of Balke is hard to reconcile, as this investigator has prepared several metallic salts of antipeptone which corresponds to Sieg- fried's formula for carnic acid. As we are not Justified in doubting the reliability of either investigator we can possibly seek the contradictory statements in the manner of procedure of the two investigators. Balke allowed the digestion to go on for only four days, while Kutscher, on the contrary, allowed it to continue for forty days; and as Kutscuer, in a sub- sequent work,' has shown that by sufficiently energetic and continuous trypsin digestion the antipeptone (the substance which gives the biuret reaction) is completely decomposed or exists only as traces, it is possible that Kutscher
' SclilitZLMiberger, Bull, de la soc. chimique de Paris, 23 ; Kuhue, Yeihandl. d. naturhist. Vereius zu Heidelberg (N. F), Bd. 1; and Klihiic aud Chittenden, Zeitscbr. f. Biologie. Bd. 19. See also Paal, Ber. d. deutscb. cbem. Gesellscb., Bd. 27.
« Zeitscbr. f. pbysiol. Cbem., Bd. 25, S. 195, aud Bd. 26, S. 110.
* Die Endprodukte der Trypsiuverdauuug, Habilitationsscbiift, Strassburg, 1899.
36 THE PROTEIN SUBSTANCES.
in his lengthy digestion experiments split the chief part of Balke's anti- peptone. This qiTestion requires further elucidation. On account of obserrations given in the previous memoir Kutscher is of the opinion that at least in the proteids of the pancreas gland the occurrence of an anti group may be excluded. He also, for other reasons, differs from the common view of Kuhne, in regard to the digestive cleavage of proteids. According to him it would be simplest and best to return to the old nomen- clature and call the primary albumoses propeptone and the deuteroalbnmoses and Kuhne's j^eptone, on the contrary, peptone.
KuHNE and his joupils, who have conducted these complete investiga- tions on the albumoses and peptones, classify the various albumoses accord- ing to their different solubilities and precipitation powers. In the pepsin digestion of fibrin' they obtained the following albumoses : {a) Hetero- albnmose, insoluble in water but soluble in dilute salt solution; (5) Fj-otalbumose, soluble in salt solution and water. These two albumoses are precipitated by ISTaCl in neutral solutions, but not completely. Hetero- albumose may, by being in contact with water for a long time or by drying, be converted into a modification, called (c) Dysalhumose, which is insoluble in dpiite salt solutions, [d) Deuieroalhumose is an albumose which is soluble is water and dilute salt solution and which is incompletely precipi- tated from acid solution by saturating with NaCl and not precipitated from neutral solutions. This precipitate is a combination of the albumose with acid (Herth°). The he teroalbumose is essentially the same, as described by Brucke, as peptone.
The albumoses obtained from different proteid bodies do not seem to be
identical, but differ in their behavior to precipitants. Special names have
been given to these various albumoses according to the mother-proteid,
namely, glohuloses^ viteUoses, caseoses, myosinoses, etc. These various
albumoses are further distinguished, a,s proto-, hereto-, and deutero -caseoses
for example. All the albumoses formed in the digestion of animal and
vegetable proteid are embraced in the common name proteoses by Chitten-
DEX.^ Certain proteoses have also been obtained in a crystalline state
(Sciirotter).
Neumeister* designates as atmidalbumose that body which is obtained by the action of siiperlieated steam on fibrin. At the same time he also obtained a substance called atmidalbuvdii, Avhich stands between the albuminates and the albumoses.
* See Kuhne and Chittenden, Zeitschr. f. Biologic, Bd. 20. ' Monatshefte f. Chem., Bd. 5.
2 Kiihne and Chittenden, Zeitschr. f. Biologic, Bdd. 22 and 25 ; Neumelster, ibid., Bd. 23; Chittenden and Ilartwell, Journ. of Phy,siol., Vols. 11 and 12 ; Chittenden and Painter, Studies from the Laboratory, etc., Yale University, Vol. 2, New Haven, 1891 ; Chittenden, iWrf., Vol. 3; Sebelien, Chem. Ceutralblatt, 1890; Chittenden and Good- win, Journ. of Physiol., Vol. 12.
* Zeitschr. f. Biologic. Bd. 26. See also Chittenden and Meara, Jourii, of Physiol., Vol. 15, and Salkowski, Zeitschr. f. Biologic, Bd. 34.
ALBUMOSES AND PEPTONES. 37
Of the soloble albnmoses Neumeister designates protoalbumose and heteroalbumose as j)n'7>iarij albnmoses, while the denteroalbnmoses, wliich are closely allied to the pej)tones, he calls secondary albionoscs. As essen- tial dilTerence between the primary and secondary albumoses he suggests the following: ' Tlfe primary albumoses are precipitated by nitric acid in salt- free solutions, wliile the secondary albumoses are only precipitated in salt solutions, and certain deuteroalbumoses, such as deuterovitellose and den- teromyosinose, are only precipitated by nitric acid in solutions saturated with NaCl. The primary albumoses are preci2)itated from neutral solutions by copper sulphate solution (2 : 100), also by XaCl in substance, while the secondary albnmoses are not. The primary albumoses are completely pre- cipitated from tlieir solution saturated with XaCl by the addition of acetic acid saturated with salt, while the secondary albumoses are only partly precipitated. The primary albumoses are readily precipitated by acetic acid and i)otassium ferrocyanide, while the secondary are only incomjiletely precipitated after some time. The primary albumoses are also, according to Pick," completely precijiitated by ammonium sulphate (add to one half saturation), while the secondary albumoses remain in solution.
The true peptones are exceedingly hygroscopic, and when perfectly dry sizzle like phosphoric anhydride when treated with water. They are exceedingly soluble in water, diffuse more readily than the albumoses, and are not precipitated by ammonium sulphate. In contradistinction to the albnmoses the true peptones are not precipitated by nitric acid (even in solution saturated with salt), by acetic acid saturated with salt and sodium chloride, potassium ferrocyanide and acetic acid, picric acid, trichloracetic acid, mercuric-potassium iodide and hydrochloric acid. They arc precipi- tated by phospho-tungstic acid, phospho-molybdic acid, corrosive sublimate (in the absence of neutral salts), absolute alcohol and tannic acid, but the precipitate may redissolve on the addition of an excess of the precipitant. As important difference between ampho-peptone and antipeptone we must also mention that the first gives Millox's reaction while the antipeptone does not.
Ill regard to the precipitation by alcohol we must call attention to the observations of Fkankel that not only are the acid coinbinaiious of peptone (Paal) soluble in alcohol, biU abo the free peptone, and Frankel has even suggested a method of preparation based on this behavior. ScniioTTER* has also prepared crystalline albumoses which were soluble in hot alcohol, especially methyl alcohol.
According to the ordinary view the albnmoses are intermediary steps in the formation of peptone, and indeed that from the primary albumoses the deuteroalbumose is derived and from thi*s then the peptone. In opposition
' Neumelster, Zeitschr. f. Biologie, Bdd. 24, 26. ' Zeitschr. f. physiol. Chem., Bd. 24.
' Frilnkel, Zur Keuntnisse der Zerfallsprodukte des Eiweisses bci peptischer und tryptischer Verdauung. Wien, 1896 ;— SchrOtter, Monatshefte f. Chem., Bdd. 14, 16.
38 TEE PROTEIN SUBSTANCES.
to this view it seems remarkable that, as found by Kuhne/ tlie deutero- fibrinoses diffnse less readily than the protofibrinoses, and also, according to Sabanejew, the denteroalbumoses have a higher molecular weight (3200) than the protalbumoses (2467-2643). The peptones have a lower molecular weight, as shown by Sabanejew, Paal, Sjoqvist,^ to lie between 400 and 250 for various preparations, Schrottee found the molecular weight of his albumoses to be 600-700. According to Paal the acid-combining power of the hydration products produced in peptonization increases as the molecular weight decreases. Cohkheim ' found this statement true, as he discovered that the antipeptone had a much higher hydrochloric acid-com- bining power than the albumoses. He also found that the heteroalbumose united with a much greater quantity of acid than the deuteroalbumose.
ScHROTTER'* objects to the above view as to the albumoses being intermediary steps in the formation of peptone, inasmuch as, according to him, no albumoses are first formed by tlie action of acids on proteids which then yield peptone, but the proteid is simul- taneously split into albumoses and peptones.
As above stated, we consider the behavior to ammonium sulphate as the absolute difference between albumoses and peptones. It is still doubtful whethei" the behavior of a single salt, the ammonium sulphate, yields suffi- <;ient basis for the characterization of two groups of albuminous bodies, the albumoses and peptones; and this question is warranted since, according to ^eumetster, we have a deuteroalbumose (formed from the protalbumose in peptic digestion) which is not comjjletely precipitated by ammonium sulphate. It seems that the transformation of proteids into peptones takes place through a number of intermediate steps similar to the trans- formation of starch into sugar through a series of dextrins, and as ammonium sulphate is not a means of separation between dextrins and sugar, although it precipitates certain dextrins, but not all, so also it is a question whether it can serve as a means of separation for the albumoses .and peptones. A complete separation of these several intermediate products, as well as their purification, is such an extremely difficult task that it is nearly impossible at present to say how far such a differentiation is warranted or feasible.
In recent times other points of difference between the peptones and albumoses has been sought for, and SciiKOTTEUaiul Frankei. ^ consider the sulphur as buch. Sciiuotter des- ignates the following as the difference between albumoses and peptones. The albumoses <;ontain more nitrogen and have a higiier moiefular weight and contain sulpiiur. Ac- cording to Fra'nkel the peptones are always free from sulphur. The albumoses, on the •contrary, contain sulphur, and lie has only found one aibumose (in Kuhne's sense) which cdid not contain sulphur.
' Zeitsclir. f. Biologic, Bd. 29.
' Sabanejew, Ber. d. deutsch. chem. Gesellsch., Bd. 26; Paal, ibid., Bd. 27; Sjo- .qvist, Skand. Arch. f. Physiol., Bd. 5.
* Paal, 1. c. ; Cohnheim, Zeitschr. f. Biologie, Bd. 33.
* Monatshefte f. Chem., Bd. 16. » Schrijtter, 1. c. ; Frankel, 1. c.
ALB U MOSES AND PEPTONES. 39
The question as to the difference between albnmoses and peptones has lately taken another phase, as it is a question whether the so-called pure peptones are true proteids or not. According to the researches of Siegfried and his pupils,' antipeptone is identical with carnic acid (see page 43). If this is true, then antipeptone is a monobasic acid with the formula C,„Il,jN,0, , having a still smaller molecular weight than the protaniins, which can hardly be considered as proteid. Under such circum- stances it seems perhaps best to drop the name antipeptone if we continue to designate such bodies pejitones, which are still true proteids (in ordinary sense). In the sufficiently energetic trypsin digestion no peptone at all is produced only simpler cleavage products, and the so-called amphopeptone formed in pepsin digestion is the only one which remains, the careful study of which will be of the greatest interest.
Lawrow'^ has recently published his investigations on the peptic and tryptic digestive products. These observations show that the products not precipitated by ammonium sulphate are not true proteids, but consist of a mixture of decomposition products of true proteids. The action of various albumoses and peptones, as also antialbumid, as well as gelatoses and gelatin peptone, upon the blood-pressure, blood-coagulation, etc., has been studied by Chittenden ' and his pupils, and in connection with this work they also ^Ive a few chemical investigations as to the questionable bodies. An antipeptone which was prepared from pure antialbumid by trypsin digestion contained on an average C 50.93; N 13,58; and S 1.62^. The low per- centage of nitrogen indicates that the body was not contaminated by basic substances, or only to an insignificant extent. On cleavage by boiling with 20^ hydrochloric acid and then determining the total nitrogen, the ammonia nitrogen, and tlie basic nitrogen contained in the phospho-tungstic acid precipitate, they found that the basic nitrogen amounted to 17.2^ of the total nitrogen of antialbumid, 27.9,^ of the hemialbumose, and 20.7^ of the hemipeptone.
What relationship do the albumoses and peptones bear to the proteid from which they are formed? The numerous analyses of different albumoses made thus far show chiefly that, with the exception of those albumoses which stand closest to the true peptones, there is no essential difference between the composition of the original proteids and the corresponding albumoses. The pure peptones, as well as certain albumoses standing close to the pure peptones, seem, on the contrary, to contain about the same amount of hydrogen and nitrogen and to be habitually poorer in carbon than the primary albumoses or the proteid.*
' See foot-note on carnic acid, foot-note 2, page 43
» Zeitschr. f. pliysiol. Chem., Bd. 26.
' Amer. Journ. of Physiol., Vol. 2.
* Elementary analyses of albumoses and peptones will be found in the works of
40 THE PROTEIN SUBSTANCES.
The elementary analyses made up to the present time have not given ns a positive answer in regard to the relationship existing between the proteids on one side and the albnmoses and peptones on the other. The view that the peptone formation is a hydrolytic splitting is accepted by Hoppe- Seyler, Kuhxe, HenninCtEK, and indeed by nearly all recent investi- gators. In support of this view we have the observations of Henninger and Hofmeister/ according to which peptones (the albnmoses) are con- verted into a proteid similar to albuminates by the action of acetic acid anhydride, or by heating so that water is expelled. According to ScHROTTER^ the albumoscs do not yield a regenerated proteid with acetic anhydride, but an acetyl defivative insoluble in water. An albaminate-like proteid may undoubtedly also be regained on heating, which is in accord with Neumeister's observations.
According to other investigators, as Maly, Herth, Lokw, and others, tlie formation of peptone is a depolymerizaiion of the proteid. A third view is that proteids and peptones are isomeric bodies; while a fourth view (Gkiessmayer ') claims that the pro- teids consist of micell groups which on peptonization are first converted into micelli and then further into molecules. Though an ordinary proteid solution contains micelli or micell bonds, so also a peptone solution contains proteid molecules.
Th^ preparation of different albumoses in a perfectly pure form is very troubWsome and accompanied with a great many difficulties. For this reason there will be given here only the general methods by which the different albumose precipitates are obtained. If we proceed from a solution of fibrin in pepsin hydrochloric acid, we first remove the syntonin or some coagulable proteid present by first neutralizing and then coagulating by heat. The neutral filtrate is saturated with jSTaCl, which precipitates a mixture of primary albumoses. This precipitate is washed with a saturated NaCl solution, pressed and dissolved in dilute salt solution. An insoluble residue remains, which is called dysalbumose. The solution of the primary albumoses is repeatedly and completely dialyzed. Heteroalbumose separates out, while the protalbumose remains in solution and may be precipitated by alcohol. Tlie above filtrate, which has had the primary albumoses removed and saturated Avith NaCl, is treated with acetic acid, which has previously been saturated with NaCl, until no further precipitate occurs. This pre- cipitate, which consists of a mixture of primary and secondary albumoses, is filtered off, the filtrate freed from salt by dialysis, and the deuteroalbu- mose precipitated by ammonium ^mlphate. The various albumoses may also be precipitated from the original solution by ammonium sulphate, dissolved in Avater and freed from ammonium sulphate by means of dialysis, and then separated as above described.
Kliline and Chittenden, cited in foot-note, page 36; also by Ilerth, Zeitschr. f. physiol. Cbcm., Bd. 1, and Monatshefte f. Chem., Bd. 5; Maly, Ptluger's Arch., Bdd. 9, 13. Ilenninger, Compt. rend., Tome 86 ; Scbrotter, 1. c; Paal, 1. c.
' Hoppe-Seyler, Physiol. Chem., Berlin, 1881 ; Klihue, 1. c. ; Henninger, 1. c. ; Ilof- meister, Zeitsclir. f. physiol. Chem., Bd. 2.
' Monatshefte f. Chem., Bd. 17.
' Maly, 1. c; Herth, 1, c; Loew, Pfluger's Arch., Bd. 31 ; Griessmayer, see Maly's Jabresb.. Bd. 14, S. 26.
SEPARATION OF CLEAVAGE PRODUCTS. 41
In the separation of primary albnmoses from the secondary, as well as in the separation of the dilTerent deuteroalbumoses, we can make use of frac- tional precipitation with ainmonium sulphate as suggested by Pick, Umijer ' has investigated the proteid-like cleavage products obtained on the pepsin digestion of ovalLnmin, seralbumin, and serglobulin by Pick's method. F, Alexander' has done the same for casein. The usefulness of this method has been established, and though certain dilTerences of the various proteids appear, still we always obtain an equal number of cleavage products, which may be separated by fractional precipitation with ammonium sulphate. The first fraction contains the primary albumoses, the second, third, and fourth fractions the various deuteroalbumoses, and the fifth and sixth two different jieptones. Casein gave only very little lieteroalbumose and then a peptone. S, Fkankel,' in the preparation of pure deutero- albumoses, first removes the primary albumoses by precipitation with copper sulphate. ^ICller^ separates the albumoses from the j^eptones by the addition of an equal volume of a 30^ ferric chloride solution and the addition of alkali until the reaction is only faintly acid. The filtrate from the voluminous precipitate is treated with zinc carbonate and filtered after thorough stirring. The filtrate is generally free from albumoses. Only in solutions of AVitte's peptone was it necessary to concentrate the filtrate to 4~i its volume and adding a little more ferric chloride and zinc carbonate to free the solution from remaining traces of albumoses.
In the preparation of true peptone we make use of a prolonged pepsin digestion, but much quicker results are obtained by the use of trypsin digestion. The albumoses must be entirely removed, which is done by alternately precipitating in acid, neutral and alkaline solution, witli ammonium sulphate. According to Kuhxe " we proceed in the following way: The sufficiently dilute and neutral solution (free from albuminates and coagulable proteids) is first precipitated, while boiling hot, with ammonium sulphate. On cooling the precijiitated albumoses and crystal- lized salt are removed by filtration and the filtrate heated to boiling, made strongly alkaline with ammonia and ammonium carbonate, again saturated with ammonium sulphate at tiie boiling temperature. Remove precipitate by filtration when cold, heat the filtrate again until all odor of ammonia is expelled, saturate with ammonium sulphate while hot, and acidify with acetic acid and filter on cooling.
The filtrate is freed from a great part of the salt by strongly concentrate ing the liquid, allowing it to cool, and removing the salt by filtration. Another large portion of the salt may be removed from this filtrate by the careful fractional precipitation with alcohol, which yields an alcoholic solu-
■• Zeitsclir. f. pbysiol. Chcni., Bd. 25.
^ Jbitl, Bd. 25, S. 411.
3 Pick, 1. c; Friinkel. Monatslieftc f. Clieru., Bd. 18.
•» Zeitscbr. f. physiol. Chem.. Bd. 26.
' Zeitschr. f. Biologic, Bd. 29.
42 TUE PROTEIN SUBSTANCES.
tion rich in peptone with only a small quantity of ammonium salt. This solution is boiled to remove the alcohol, and then boiled with barium car- bonate to remove the ammonium sulphate. The filtrate is freed from excess of barium by the careful addition of dilute sulphuric acid. This filtrate, which must not contain an excess of sulphuric acid, is now concen- trated and the peptone precipitated therefrom by alcohol.
Frankel has suggested another method which is dependent upon the solubility of the peptones in alcohol. Baumann and Bomer ' precipitate the albumoses by zinc sulphate.
For the detection of albumoses and peptones in animal fluids we proceed as follows, according to Devoto : The coagulable proteids are removed by prolonged heating, the solution saturated with ammonium sulphate. True peptones (besides deuteroalbumose not precipitated) may be detected in the cold filtrate by means of the biuret test. The remaining albumoses are contained in the mixture of precipitate and salt crystals collected on the filter. The albumoses are dissolved from this mixture by washing with water, and may be detected in the wash-water by means of the biuret test. According to Halliburton and Colls" traces of albumoses may be formed in this method by the prolonged heating. As the best method they suggest either the precipitation of the native proteids by the addition of IQfo trichloracetic acid solution or making the native proteids insolnble by the )^ntinuous action of alcohol. The last method is not quite applicable to blood-serum, as the so-called fibrin-ferment, which also gives the biuret test, is not made insolnble by this procedure.
If a solation saturated with ammonium sulphate is to be tested by the biuret test, it must first be treated with a slight excess of concentrated caustic-soda solution, keeping the solation cold, and after the sodium sulphate has settled the liquid is treated with a 2^ solution of copper sulphate, drop by drop.
The biuret test (colorimetric) and the polariscopic method have been used in the quantitative estimation of albumoses and peptones. These methods do not yield exact results.
Coagulated Proteids. Proteids may be converted into the coagulated condition by different means: by heating (see page 25), by the action of alcohol, especially in the presence of neutral salts, by prolonged shaking their solutions (Ramsdex'), and in certain cases, as in the conversion of fibrinogen into fibrin (Chapter VI), by the action of an enzyme. The nature of the processes which take place during coagulation is unknown. The coagulated albuminous bodies are insoluble in water, in neutral salt solutions, and in dilute acids or alkalies, at normal temperature. They are dissolved and converted into albuminates by the action of less dilute acids or alkalies, especially on heating.
Coagulated proteids seem also to occur in animal tissues. We find, at
' Frilnkel, 1. c, Zur Kenntniss, etc.; Bomer, Cliem. Centralbl. 1898, 1, S. 640. « Devoto, Zeitschr. f. physiol. Clieni., Bd. 15 ; Halliburton and Colls, Journ. of Path. andBact., 1895.
' Du Bois-Reymoud's Arch., 1894.
VEGETABLE AND POISONOUS PR0TETD8. 43
least in many organs such as the liver and other glands, proteids wliich are not soluble in water, dilute salt solutions, or very dilute alkalies, and only dissolve after being modified by strong alkalies.
Appendix.
Vegetable Proteids. A'egetable proteids seem to have the same essential properlieri as the animal proteids, and the three chief groups of native proteids occur in the plants as well as the animal organism. We recognize the following as vegetable proteids: albumi)is^ glohulins (phytovitellin, vegetable myosin, paraglobulin), and micleoalbmnitis (pea-legumin). Be- sides these a special group of coagulated proteids, so-called gluten proteins, occur, which are partly soluble in alcohol. It seems that too much im- portance is given to the solubilities of the vegetable proteids, and more exhaustive investigations seem to be necessary.'
Poisonous Proteids. Attention was called in the first chapter to the fact that high i)lants and animals, as well as microbes, can produce proteids having specific, sometimes intense, poisonous action.
We know very little positively in regard to the nature of these proteids. Those which have been isolated belong to certain of the proteid groups — some are albumins, others globulins or compound proteids, and the majority seem to be albumoses — still little is known in regard to their chemical nature. From a chemical standpoint we do not differentiate between a poisonous and a harmless proteid; for example, between a poisonous and a non-poisonous globulin. The fundamental question whether those that have been isolated as poisonous proteids are really poisonous or not, oi whether they consist of a harmless proteid contaminated with a polsonou? substance, cannot be considered as settled.
Carnic acid, which is considered as identical with antipeptone, stands h close relationship to the so-called true peptones.
Carnic Acid. This acid, discovered by Siegfried, was first obtaineLi tvs a c eivage product of phospho-carnic acid occurring in muscles (see Chapter XI). Carnic acid is produced from the proteid, according to Siegfried, under the same conditions as antipeptone, with which Balke' considers it identical (see page 35). It is a monobasic acid with the formula CjgHijNgOj. It is split into lysin, lysatin, and ammonia by 15^ hydro- chloric acid at 130° C, which seems remarkable when we consider the low molecular weight of the acid and the presence of only three atoms of
' See Kjeldabl : Undersogelser over de optiske Forhold lios nogle PlanteiCggelivide- stoffer. Forhaudlingcr vqd de skaudinaviske Xalurfoiskeies 14. M5de. KjObcn- bavn, 1892.
' Siegfried, Du Bois-Reymond's Arch., 1894, and Zeitschr. f. pbysiol. Cbem., Bd. 21 ; Balke, ibid., Bd. 22.
44 THE PROTEIN SUBSTANCES.
nitrogen in the molecule. On the oxidation of the barium salt by barium permanganate oxycarnic acid, with the formula, Cg^H^jNgOjj , is obtained, which is derived from three molecules of carnic acid with the elimination of four atoms of hydrogen.
Carnic acid is an extremely hygroscopic substance, being very sol able in water. It also dissolves in hot alcohol and separates out as undefined crystalline plates on cooling. It gives with hydrochloric acid an additional product with the formula Cj^Hj^lSTjOj-HCl, and also yields salts with several metals. Among the salts the silver salt with 42.G;o silver is of special importance. This acid acts like antipeptone towards most precipitants and, like this, is not precipitated by ammonium sulphate.
Tbe methods of preparing caruic acid from proteids are the same as tlie methods of preparing pure untipeptones in tryptic digestion. According to Siegfried carnic acid is obtained from meat extract iu the following way: The extract free from proteids is com- l)lftely precipitated with calcium chloride and ammonia. The phosphocarnic acid is precipitated from ihe filtrate as au iron combination, carniferrin, bj^ ferric chloride. This carniferrin is decomposed at 50° by barium hydrate, filtered, the excess of barium re- moved from the filtrate by sulphuric acid, filtered, concentrated and precipitated with alcohol. The acid is purified by repeated resolution and precipitation with alcohol.
/ II. Com pound Proteitls.
With this name 77e designate a class of bodies which are more complex than the simj^le proteids and which yield as nearest splitting products simple proteids on one side and non-proteid bodies, such as coloring matters, carbohydrates, xanthin bodies, etc., on the other.'
The compound proteids known at the present time are divided into three chief groups. These groups are the hcmnoglohins, the glycoproteids, and the nndeojyroteids. The hsemoglobins Avill be treated of in a following chapter (Chapter YI), on the blood.
Glycoproteids are those compound proteids which on decomposition yield a proteid on one side and a carbohydrate or derivatives of the same on the other, but no xanthin bodies. Some glycoproteids are free from phos- phorus (mucin substances, chondroproteids, and hyalogens), and some contain phosphorus (phosphoglycoproteids).
Mucin Substances. We designate as mucins colloid substances whose solutions are mucilaginous and thready, and wliich when treated with acetic axjid give a precipitate insoluble in an excess of acid, and on boiling with dilute mineral acids yield a substance capable of reducing copper oxyhydrate. This last-mentioned fact, which was first observed by EicinvALD,° differen- tiates mucins from other bodies which have long been mistaken for it and which have similar physical properties. On the other hand, bodies whose
' Hoppe-Seyler lias given tlie name proie'ide to these compound proteids, but as this term is misleading in Engli.sh we do not use it iu English classifications in this sense, ' Annal. d. Chem. u. Pharni., Bd. 134.
MUCINS. 45
pliysical properties dilTer from it, but wliich give a reducible substance on boiling with dilute mineral acids, liave also l)een designated as mucins.
The ditTerent bodies characterized as mucin substances correspond, first, either to true vmcins, or, second, to mucoids or mucifwidSy or third to vhondruprufcids.
All mucin substances contain carbon, hydrogen, nitrogen, sulphur, and v.rggen. Compared with albuminous bodies they contain less nitrogen and, as a rule, considerably less carbon. As immediate decomposition products they yield albuminous bodies on one side and carboliydrates or acids allied thereto on the other. On boiling with dilute mineral acids they all give a reducing substance.
The true mucins are characterized by their natural solution, or one prepared by the aid of a trace of alkali, being mucilaginous, thread-like, and giving a precipitate with acetic acid which is insoluble in excess of acid. The mucoids do not show these physical properties and have other solubili- ties and precipitation properties. As we have intermediate steps between different albuminous bodies, so also we have such between true mucins and mucoids, and a sharp line between these two groups cannot be drawn.
True mucins are secreted by the larger mucous glands, by certain mucous membranes, also by the skin of snails and other animals. True mucin also occurs in the connective tissue and navel-cord. Sometimes, as in snails and in the membrane of the frog-egg (Giacosa '), a mother- substance of mucin, a mucinogen, has been found which may be converted into mucin by alkalies. Mucoid substances are found in cartilage, certain cysts, in the cornea, the crystalline lens, white of egg, and in certain ascitic fluids. As the mucin question has been very little studied, it is at the present time impossible to give any positive statements in regard to the occurrence of mucins and mucoids, especially as without doubt in many cases non-mucinous substances have been described as mucins. So mucli is sure, that mucins or nearly related bodies occur widely diffused in the organism in certain tissues. From their decomposition products we derive a great deal of knowledge in regard to the formation and cleavage of carbo- hydrates or kindred l)odies (glycuronic acid) from other complex groups.
True Mucins. Thus far we have been able to obtain only a few mucins in a pure and nnclianged condition due to the reagents used. The elemen- tary analyses of these mucins have given the following results:
C H N S O
Mucin from snail 50.32 6.84 i:3.65 1.75 27.44 (Hammarsten)
Mucin from tendon 48.30 6.44 11.75 0.81 32.70 (Loebisch)
Mucin from submaxillary. .. 48.84 6.80 12.32 0.84 31.20 (Hammaksten)
The mucin of the snail-skin, which stands closest to keratin, contains more sulphur than the other mucins. The same is true for the mucin
' ZeitscUr. f. pbysiol. Chem., Bd. 7 ; also Hammarsteu, Pflliger's Arcbiv, Bd. 36-
46 TEE PROTEIN SUBSTANCES.
obtained from the Achilles tendon of oxen as prepared by Chittenden and GiEs/ which contains on an average 2.33^ snlphar. The sulphur is, at least in certain mucins, partly split oif by alkali, and in others not.
By the action of superheated steam on mucin a carbohydrate, animal gnm (Landwehk), is split off. This has not been substantiated by other investigators such as Folin and F. Muller.''' Instead of a non-nitrogenous gum a nitrogenous carbohydrate was obtained.
On boiling mucin with dilute mineral acids, acid albuminate and bodies similar to albumose or peptone are obtained, besides a reducing substance. MuLLER obtained 25-32^ reducing substance on boiling the mucus from the respiratory organs with "di sulphuric acid. He also prepared a crystal- line phenylhydrazine combination therefrom having a melting-point of 198° C. and differing in other regards from glucosazon. He considers it as an osazon of a hexose which he calls imicose. Muller could not prepare the sugar itself, but obtained a crystalline substance containing 6.4^ N and considered as mucosamin. Jazewitz ^ could not obtain any sugar from mucin but an osazon melting at 185° C. and a mucosamin. Muller^ by a different and better method has obtained a benzoyl combination, and then frorn this a crystalline hydrochloric acid combination of its mucosamin, by boiling mucin with acids. The crystallographic researches, as well as the determination of its optical rotation, show so much to the identity of this combination with chitasamin hydrochloride that Muller considers the name mucosamin unnecessary. The osazon obtained from this combination differs, on the contrary, from the glucosazon in the following: It melts at 192 to 196°, it is readily soluble in alcohol, and is la3vo-rotatory. According to E. Fischer, who has investigated it, it is not identical with glucosazon, but seems rather to be galactosazon. On boiling mucins with hydrochloric acid acetic acid may also be sjilit off, and indeed ^-1 molecule for each molecule of reducing substance. By the action of stronger acids we obtain among other bodies leucin, tyrosin, and levulinic acid. Certain mucins, as the submaxillary mucin, are easily changed by very dilute alkalies, as lime- water, while others, such as tendon-mucin, are not affected. If a strong caustic-alkali solution, as a bfo KOH solution, is allowed to act on submaxil- lary mucin, we obtain alkali albuminate, bodies similar to albumose and peptone, and one or more substances of an acid reaction and with strong reducing powers.
' Hammarsteii, Pfliiger's Arch., Bd. 36, aiul Zeitschr. f. ph3'siol, Chem., Bd. 12 ; Loebisch, ibid., Bd. 10, and Chittenden and Gies, Journ. of Expt. Med., Vol. 1.
» Landvvelir, Zeitschr. f. physiol. Chem., Bdd. 8, 9 ; also Pfluger's Arch., Bdd. 39 and 40 ; Folin, Zeitschr. f. physiol. Chem., T.d. 28; Fr. Miiller, Sitzungsber. d. Gesell- Bch. zur BefOrd. d. gcsamnit. Naturwiss. zu Marburg, 1896.
» Mailer, 1. c; Jazewitz, Arch. d. scien. bid. do St. Petersbourg, Tome 5.
* Sitzungsber. zur BefOrd. d. gesammt. Naturwiss. zu Marburg, 1898.
MUCOTDS. 47
In one or the other respect the dillerent mneins act somewhat differently. For examj)le, the siuiil and tendon mucins are insoluble in dilute liydro- chloric acid of \-l p. m., while the mucin of the submaxillary gland and the navel-cord are soluble. Tendon-mucin becomes llaky with acetic acid, while the other mucins are precipitated in more or less fibrous, tough masses. Still all the mucins have certain reactions in common.
In the dry state mucin forms a white or yellowish-gray powder. When moist it forms, on tlie contrary, ilakes or yellowish-white tough lumps or masses. The mucins are acid in reaction. They give the color reactions of the albuminous bodies. They are not soluble in water, but may give a neutral solution with water and the smallest quantity of alkali. Such a solution does not coagulate on boiling, while acetic acid gives at the normal temperature a precipitate which is insoluble in an excess of the precipitant. If 5-10^ XaCl be added to a mucin solution, this can now be carefully acidified with acetic acid without giving a precipitate. Such acidified solu- tions are copiously precijiitated by tannic acid; Avith potassium ferrocyanide they give no precipitate, but on sufiicient concentration they become thick or viscous. A neutral solution of mucin-alkuli is precipitated by alcohol in the presence of neutral salts; it is also precipitated by several metallic salts. If mucin is heated on the water-bath with dilute liydrochloric acid of about 2^, the liquid gradually becomes a yellowish or dark brown and reduces copper oxyhydrate from alkaline solutions.
The mucin most readily obtained in large quantities is the submaxillary mucin, which maybe prepared in the following way: The filtered watery extract of the gland, free from form-elements and as colorless as possible, is treated with 25^ hydrochloric acid, so that the liquid contains 1.5 p. m, HCl. On the addition of the acid the mucin is immediately precipitated, but dissolves on stirring. If this acid liquid is immediately diluted with 2-3 vols, of water, the mucin separates and may be purified by redissolving in 1-5 p. m. acid, and diluting with water and washing therewith. The mucin of the navel-cord may be prepared in the same way.^ The tendon- mucin is prepared from tendons which have first been freed from proteid by common-salt solution and water. They are extracted with one half saturated lime-water, the filtrate is precipitated with acetic acid, and the precipitate purified by redissolving in dilute alkali or lime-water, precipitat- ing with acid, and washing with water (Rollett, Loebisch, Chittenden", and GiEs).' Lastly, the mucins are treated with alcohol and ether.
Mucoids or Mucinoids. In this group we must include those non- phosphorized glycoproteids which are neither true mucins nor chondro- proteids even though they show amongst themselves such a difference in behavior that they can be divided into several sub-groups of mucinoids. To the mucinoids belong pseudoimicin, the probably related body colloid,
' The author has not been able to obtaiu this pure, so the aiialyis is uot given in the previous table of the mucins.
» Rollett, Wieu. Sitzungsber., Bd. 39, Abth. 2 ; Loebisch, Chittenden and Gies, 1. c.
48 TEE PROTEIN SUBSTANCES.
ovomucoid^ and other bodies, which on account of their differences will be
best treated of individually in their respective chapters.
Hyalogens. Under this name Khukenberg' Las designated a number of differing bodie's, wiiicb are characterized by the following : By the action of alkalies they change, with the splitting off of sulphur and some nitrogen, into soluble nitrogenized products called by him hyalines and which yield a pure carbohydrate by further decomposition. We lind that very heterogeneous substances are included in these groups. Certain of these hyalogens seem undoubtedly to be glycoproteids. Neossin ■ of the Chinese edible swallow's-nest, membranin ^ of Debcemet's membrane and of the capsule of the cr3^stalline lens, and spirogra'pldn * of the skeletal tissue of the worm Spirographis seem to act as such. Others on the contrary, such as hyalin ^ of the walls of hydatid cysts, oniipldn^ from the tubes of Ouuphis tubicola, seem not to be compound proteids. The so-called mucin of the holoihures,'^ and chondrosin^ of the sponge, Chondrosia reui- formis. and others may also be classed Avith the hyalogens. As the various bodies desig- nated by KiiUKENBERG as hyalogens are very dissimilar, it is not of much importance to arrange these in special groups.
Chondroproteids are such glycoproteids which as closest cleavage products yield proteid and an ethereal sulphuric acid containing carbo- hydrate, cliondroitin-sulphuric acid. Chondromucoid, occurring in cartilage is the best example of this group. Amyloid occurring under pathological conditions also belongs to this group. On account of the property of chondroitin-sulphuric acid of precipitating proteid it is also possible that under certain circumstances combinations of this acid with proteid may be precipitated from the urine and be considered as chondroproteids.
Chondromucoid has greatest interest as a constituent of cartilage, and on this account this body and also its cleavage product, chondroitin-sulphuric acid, will be treated of in connection with cartilage (Chapter X). On the contrary, amyloid, which has always been treated of in connection with the protein substances, will be described here.
Amyloid, so called by Vmcuow, is a protein substance appearing under pathological conditions in the internal organs, such as the spleen, liver, and kidneys, as infiltrations; and in serous membranes as granules with con- centric layers. It probably also occurs as a constituent of certain prostate calculi. The chondroproteid occurring under physiological conditions in the walls of the arteries is perhaps, according to Krawkow, very nearly related to the amyloid substance even if not identical.
Amyloid was first prepared pure recently by Krawkow. ° The sub-
' Verb. d. physik. -med. Gesellsch. zu Wiirzburg, 1883 ; also Zeitschr. f. Biologic, Bd. 22.
2 Krukenberg, Zeitschr. f. Biologic, Bd. 22.
2 C. 'Ih. Morner, Zeitschr. f. i)liysiol. Chem., Bd. 18.
* Krukenberg, Wiirzburg, Verhandl. 1883 ; also Zeitschr. f. Biologic, Bd. 22.
' A. Lllcke, Virchow's Arch., Bd. 19 ; also Krukenberg, Vergleichende physiol. Stud., Series 1 and 2, 1881.
« Schmiedeberg, Milth. aus d. zool. Stat, zu Neapel, Bd. 3, 1882. ' Hilger, Pili'iger's Archiv, Bd. 3.
* Krukenberg, Zeitschr. f. Biologie, Bd. 22.
* Arch. f. exp. Path. u. Pharm., Bd. 40, which also contains the older literature.
AMYLOID AND PnOSPHOOLYCOPJiOTEIDS. 49
stance prepared by liim contained C 4:8.80-50.38; II (J. 0.5-7. 02; N 13.79- l-i.07; and S 2.G5-2.89f^. Phosphorus does not occur in the pure sub- stance. It splits, by the iiction of alkali, into proteid and chondroitin- sulphuric acid (see Chapter X) and according to Krawkow is therefore perhaps an ester-like combination of this acid with proteid.
Amyloid is an amorphous white substance, insoluble in water, alcohol, ether, dilute hydrochloric and acetic acids. It is soluble in concentrated hydrochloric acid or caustic alkali with decomposition. On boiling with dilute hydrochloric acid it yields sulphuric acid and a reducing substance. It is not dissolved by gastric juice. It is nevertheless changed so that it is soluble in dilute ammonia, while the genuine typical amyloid is insoluble therein. Amyloid gives the xanthoproteic reaction and the reactions of MiLLOX and Auamkiewicz. Its most important property is its behavior with certain coloring matters. It is colored reddish brown or a dingy violet by iodine; a violet or blue by iodine and sulphuric acid; red by methylaniline iodide, especially on the addition of acetic acid; and red by aniline green. Of these color reactions those with aniline dves are the most important. The iodine reaction appears less constant and is greatly dependent upon the physical condition of the amyloid. The color reactions are dependent upon the presence of the chondroitin-sulphnric acid com- ponent.
The preparation of amyloid may be performed as follows according to Kraavkow: The finely divided mass of organ is exhausted first with water and then with dilute ammonia, Avhich leaves the insoluble amyloid and removes the free or the combined chondroitin-sulphnric acid besides other substances. The product, after being washed Avith water, is digested with pepsin for several days at 38° C. The residue, after washing with hydro- chloric acid and water, is dissolved in dilute ammonia, filtered, again precipitated with dilute hydrochloric acid, dissolved, if necessary, in ammonia, precipitated a second time with hydrochloric acid, washed with water, the precipitate dissolved in baryta-water, which leaves the nucleius undissolved, and the barium filtrate precipitated with hydrochloric acid, and then washed with water, alcohol, and ether.
Phosphoglycoproteids. This group includes the pbosphorized glycopioteids. Tbey yioM no xanthiu substances (nuclein bases) as cleavage products. Tbey :ire not nucleo- l)roteids and tberefore Ibey must not be considered togetber witb the glycouuclcopro- teids (uucleoglycoprolelds) or mistaken for tbem. On pepsin digestion tbey may like certain uucleoalbumins yield pseudonuclein, but tbey dilTer from tbe nucleoalljumins in tbat tbey yield a reducing substance on boiling witb dilute acid. Tbey differ from tbe glycouucleoproteids in tbat tbey do not, as above mentioned, yield any xantbin bodies.
Only two pbospborized glycoproteids are known at tbe present time, namely, ich- thulin, occurring in carp eggs and studied by Waltek ' and wbich was considered as a vitelliu for a time. Icbthulin has tbe following composition : C 53.52; H 7.71 ; N 15.64; S 0.41 ; P 0.43; F^ 0A0%. In regard to solubilities it is similar to a globulin. Walter has prepared a reducing substance from tbe parauucleiu of icbthulin which gave a very crystalline combination with pbenylbydrazin.
' Zeitschr. f. physiol. Chem., Bd. 15.
50 THE PROTEIN SUBSTANCES.
Another phosphoglycoproteid is litlicoproteid, obtaiued by the author' from the glauds of the snail Helix pomatia. It has the following composition: C 46.99; H6.78; N 6.U8 ; S 0.62 ; P 0.47/o. It is converted into a gummy, Isevo-rotatory carbohydrate, called animal sinistrin, by the action of alkalies. On boiling with an acid it yields a dextro-rotatory, reducing substance.
Nucleoproteids. With this name we designate those compound proteids which yield trne nncleins (see Chapter V) on j)epsin digestion and those which yield, besides proteids, xanthin bodies or so-called naclein bases (parin bases) on boiling with dilate mineral acids.
The nucleoproteids seem to be widely diffused in the animal body. They occur chiefly in the cell-nuclei, but they also often occur in the proto- plasm. They may pass into the animal fluids on the destruction of the cells, hence nucleoproteids have also been found in blood-serum and other fluids.
They may be considered as combinations of a proteid nucleus with a side chain, which Kossel calls the prostetic group. This side chain, which contains the phosphorus, may be split off as nucleic acid (see Chapter Y) on treatment with alkali. As we have several nucleic acids, it follows that we must/ have different nucleoproteids, depending upon the nucleic acid united with the proteid. Certain nucleic acids contain a readily sj)lit off' sugar (pentose or hexose), others on the contrary not. In the first case we obtain from the corresponding nucleoproteid a reducing sugar on boiling with dilute mineral acid, while in the other case this is not possible. This different behavior may be accounted for by a special group of nucleoproteids, the glyconucleoproteids or nucleoglycoproteids. Such glyconucleoproteids occur in yeast-cells, in the pancreas, and, as it ajipears, are widely dis- tributed in the animal organism.
The native nucleoproteids contain a variable but not a high percentage of phosphorus, which Halliburton' found to vary between 0.5^ and 1.6^. On heating their solutions, as well as by the action of dilute acids, a modification of the compound proteid takes place and nucleoproteids of strong acid character, poorer in proteid but richer in phosphorus, are formed. The native nucleoproteids have faint acid properties and are in- soluble in water but whose alkali combinations soluble in water split on heating their solution into coagulated proteid and a nucleoproteid rich in phosphorus, which remains in solution. In peptic digestion they yield so- called true nuclein. The proteid can be precipitated by acetic acid from its alkali coml^ination, and the precipitate dissolves Avith more or less readiness in an excess of the acid. A confusion may occur here with nncleoalbumins and also with mucin substances. This confusion may be avoided by warm- ing the body for some time on the watcr-l)ath with dilute sulphuric acid, nearly neutralizing the boiling-hot fluid with barium hydrate, filtering as.
' HammarstcD, Pfinger's Arch., Bd. 36. « Journ. of Physiol., Vol. 18.
ALB V MOWS OR ALBUMINOIDS. 61
quick as possible while boiling hot, supersaturating the filtrate with am- monia, and then on cooling (when a precipitate consisting of guanin is filtered oil and specially tested) testing for xanthin bodies by an ammoniacal silver nitrate solntion. Any precipitate formed is examined more closely by the method as given in Chapter \ . The nncleoproteids give the color reac- tions of the proteids.
The properties of the various nncleoproteids are given in detail in the various chapters which follow.
III. Albumoids or Albuiiiiiioids.
Under this name we collect into a special group all those protein bodies which cannot be placed in either of the other two groups, although they differ essentially among themselves and from a chemical standpoint do not show any radical difference from the true proteid bodies. The most im- portant and abundant of the bodies belonging to this group are important constituents of the animal skeleton or the cutaneous structure. They occur as a rule in an insoluble state in the organism, and they are distinguished in most cases by a pronounced resistance to reagents which dissolve proteids, or to chemical reagents in general.
The Keratin Group. Keratin is the chief constituent of the horny structure, of the epidermis, of hair, wool, of the nail, hoofs, horns, feathers, of tortoise-shell, etc., etc. Keratin is also found as neurokeratin (Kuhne) in the brain and nerves. The shell-membrane of the hen's egg seems also to consist of keratin, and according to Neumeister ' the organic matrix of the egg-shells of various vertebrate animals belongs in most cases to the keratin group.
It seems that there exist more than one keratin, and these form a special group of bodies. This fact, together with the difficulty in isolating the keratin from the tissues in a pure condition without a partial decomposi- tion, is sufficient explanation for the variation in the elementary composition given below. As examples the analyses of a few tissues rich in keratin and of keratins are given as follows : '
C H N S O
Human hair... 50.65 6.36 17.14 5.00 20.85 (v. Laar)
Nail 51.00 6.94 17.51 2.80 21.75 (Mulder)
Neurokeratin.... 56.11-58.45 7.26-8.02 11.46-14.32 1.63-2.24 (Kuhne)
Horn (averas^e).. 50.86 6.94 3.30 . . . (Hohbaczewski)
Tortoise-shell.... 54.89 6.56 16.77 2.22 19.56 (Muldp:k)
Shell-membrane. 49.78 6.64 16.43 4.25 22.90 (Lindvall)
' Kilhne and Ewald, Verh. d. naturhistor.-med. Vereins zu Heidelberg (X. F.), Bd. 1 ; also Klihue and Chittenden, Zeitschr. f. Biologic, Bd. 23 ; Neumeister, ibid., Bd. 31.
* V. Laar, Anual. d. Chem. u. Pharm., Bd. 45 ; — Mulder, Versuch einer allgem. physiol. Chem., Braunschweig, 1844-51; Kiihne, Zeitschr. f. Biologic, Bd. 26; Hor- baczewski, see Drechsel in Ladenburg's HandwOrterbuch d. Chem., Bd. 3; Lindvall, Maly's Jahresbericht, 1S81.
52 THE PROTEIN SUBSTANCES.
MoHii ' has determined the quantity of sulj^hur in various keratin sub- stances. Sulphur is at least in part in loose combination, and it is partly removed by the action of alkalies (as sulphides), or indeed in part by boiling with water. Combs of lead after long usage become black, and this is due to the action of the sulphur of the hair. On heating keratin with water in sealed tubes to a temperature of 150° to 200° C. it dissolves, with the elimination of sulphuretted hydrogen, forming a non-gelatinizing liquid which contains albumose (called Iceratinose by Krukenberg ") and pep- tone (?). Keratin is dissolved by alkalies, especially on heating, forming, besides alkali sulphides, albumoses and peptones (?).
The dconiposition products of keratins are moreover the same as the true proteids. On boiling with acids we obtain besides leucin and tyrosin, which occurs in relatively great amounts (1-5^), aspartic acid^ and glutamic acid,^ ammonia, and sulphuretted hydrogen. Hedin " has obtained lysin, arginin, and a substance containing sulphur, whose combination with HCl has the composition Cj^Hg^X^O^.^SCl^ , from horn shavings.
There is no doubt that the keratins are derived from the proteids. Dre^hsel ° is also of the opinion that in the keratin a part of the oxygen of the proteids is exchanged for sulphur, and a part of the leucin, or any other amido-acid, is exchanged for tyrosin. Keratin and proteids give the same decomposition products, with the exception that the former gives proportionally a greater quantity of tyrosin. Among the sulphurized cleavage products of keratin Emmerlixg found cystin, and Suter' thio- ladic acid. Suter conld not detect either cystin or cystein.
Bodies occur in the animal kingdom which form intermediate bodies between coagulated albumin and keratin. C. Th. MoRXERMias detected such a body {alhumoid) in the tracheal cartilage, which forms a net-like trabecular tissue. This substance appears to be related to the keratins on account of its solubilities and on the quantity of the sulpuhur (which turns lead black) it contains, while according to its solubility in gastric juice it must stand close to the proteids. Another substance, more similar to keratin, forms the horny layer in the gizzard of birds. According to J. IIedenius" this substance is insoluble in gastric or pancreatic juice and
' Zeitschr. f. physiol. Cbem., Bd. 20.
* Uutersucli. liber d. cheni. Ban d. Eiweisskorper. Sitzuugsber. d. Jeuaiscbcn Gesellsch. f. Med. u. Naturwissoiiscb., 188G.
* Kreusler, Journ. f. prakt. Cbeni., Bd. 107.
* Ilorbaczcwski, Sitzungsber. d. k. k. Wieu. Akad. d. Wissensch., Bd. 80.
'> Kgl. fysiogr. Sallsk. i Lund bandlingar, Bd. 4; also Maly's Jabresber., 1893, and Zeitschr. f. physiol. Chem., Bdd. 20 and 21.
* Drecbsel in Ladenburg's Ilandwortcrbucli d. Cliem., Bd. 3.
' Emmcrliug, Bef. in Cbcmiker Zoitg., Mo. 80, 1894 ; Suter, Zeitschr. ^f. physiol, Chem.. Bd. 20.
» See Maly's Jabresber., 1888. » Skan. Arcb. f. Pbysiol.. Bd. 3.
ELA8TIN. 53
acts (juite similar to keratin. It contains only I'ji snlphnr, and yields on decomposition only very little tyrosin besides considerable leucin.
Keratin is amorphous or takes the form of the tissues from which it was prepared. On heating it decomposes and generates an odor of burnt iiorn. It is insoluble in water, alcoliol, or ether. On heating with water to 150°-200° C. it dissolves. It also dissolves gradually in caustic alkalies, especially on heating. It is not dissolved by artificial gastric juice or by trypsin solutions. Keratin gives the xanthoproteic reaction, as well as the \eaction with Millox's reagent, although not always typical.
In the preparation of keratin a finely divided horny structure is treated first with boiling water, then consecutively with diluted acid, pepsin-hydro- chloric acid, and alkaline trypsin solution, and, lastly, with water, alcohol, and ether.
Elastin occurs in the connective tissue of higher animals, sometimes ia such large fiuantities that it forms a special tissue. It occurs most abundantly in the cervical ligament (ligamentum nucha").
Elastin is generally considered as a sulphur-free substance. According to the investigations of Ciiittexdex and Hart, it is a question whether or not elastin does not contain sulphur, which is removed by the action of the alkali in its preparation. II. ScnwARZ has been able to prepare an elastin containing sulphur from the aorta by another method, and this sulphur can be removed by the action of alkalies, without changing the properties of the elastin, and recently Zoja, Hedin, and Bergh ' have found that elastin contains sulphur. The most trustworthy analyses of elastin from the cervical ligament (Xos. 1 and 2) and from the aorta (N:>. 3) have given the follow- ing results:
S O
.... 21.94 (HORBACZEWSKl)'^
21.79 (Chittenden and Haut)
0.38 (H. ScuwAKz)
Zoja found 0.270,'^ sulphur and 16.9G^ nitrogen in elastin. IIedin" and Bergh found different quantities of nitrogen in elastin, depending upon whether Horbaczewski's or Sciiavarz's method was used in its prepara- tion. In the first case they found 15.44f^ nitrogen and 0.55^ sulphur, and in the other 14.07,'^ nitrogen and O.GG^^ sulphur.
The cleavage products of elastin are the same as for the true jiroteids, with the difference that glycocoll but no aspartic and glutamic acids are obtained.' Tyrosin is only obtained in small quantities. Schwarz was able to detect lysatin in the decomposition products, but IIedix and
' Cliitteiuleu and Ilait, Zeilschr. f. Biologic, Bd. 25; Sclnvarz, Zeilscbr. f. pbysiol. Cbem., Bd. 18 ; Zoja, ibid., Bd. 23 ; Bergh, ibid., Bd. 25 ; Hediu, ibid. ' Horbaczewski, Zeitscbr. f. pbysiol. Cbem., Bd. 6. * See Drccbscl in Ladenburg's Haudworterbucb, Bd. 3.
|
C |
H |
N |
|
|
1. |
54.32 |
6.99 |
16 75 |
|
2. |
54 24 |
7.27 |
16.70 |
|
3. |
53.95 |
7.03 |
16.67 |
54 THE PROTEIN SUBSTANCES.
Bergh could not find either lysin (lysatin) or arginin. On putrefaction by anaerobic micro-organisms Zoja foand carbon dioxide, hydrogen, methane, mercaptan, butyric acid, valerianic acid, ammonia, and possibly also phenylpropionic acid and aromatic oxyacids. Indol and skatol have not been found in putrefaction,' but Schwarz, on the contrary, obtained indol, skatol, benzol, and phenols, on fusing aorta-elastin with caustic potash. On heating with water in closed vessels, on boiling with dilute acids, or by the action of proteolytic enzymes, the elastin dissolves and splits into two chief products, called by Horbaczewski hemielastin and elastin^jeptone. According to Chittenden and Hart, these products correspond to two albumoses designated by them protoelastose and deuteroelastose. The first is soluble in cold water and separates on heating, and its solution is precipi- tated by mineral acid as well as by acetic acid and potassium ferrocyanide. The watery solution of the other does not become cloudy on heating, and is not precipitated by the above-mentioned reagents.
Pure dry elastin is a yellowish-white powder; in the moist state it appears like yellowish-white threads or membranes. It is insoluble in wate^", alcohol, or ether, and shows a resistance against the action of chehiical reagents. It is not dissolved by strong caustic alkalies at the ordinary temperature, and only slowly at the boiling temperature. It is very slowly attacked by cold concentrated sulphuric acid, and it is relatively easily dissolved on warming with strong nitric acid. Elastins of differing origins act differently with cold concentrated hydrochloric acid; for in- stance, elastin from the aorta dissolves readily therein, while elastin from the ligamentum nuchas, at least from old animals, dissolves with difficulty. Elastin is more readily dissolved by warm concentrated hydrochloric acid. It responds to the xanthoproteic reaction and with Millon's reagent.
On account of its great resistance to chemical reagents, elastin may be prepared (best from the ligamentum nucha?) in the following way: First boil with water, then with 1^ caustic potash, then again Avith water, and lastly with acetic acid. The residue is treated with cold b<fo hydrochloric acid for twenty-four hours, carefully washed with water, boiled again with water, and then treated with alcohol and ether.
aSchwarz first incompletely digested the tissues with pepsin, washed first with soda solution and then with water, and boiled lastly with water until the elastic substance was dissolved away. The dried and powdered substance is again digested witli gastric juice and treated as above, and then boiled with water until the contaminating reticulin-like substance is com- pletely removed.
Collagen, or gelatin-forming substance, occurs very extensively in verte- brates. The llesh of cephalopods is claimed to contain collagen.'' Collagen
' Walcbli, Journ. f. prakt. Chem., Bd. 17.
' iloppeSeyler, Pbysiol. Chem. Beiliu, 1877-81. S. 97.
COLLAGEN. 55
is the chief constituent of the fibrils of the connective tissue and (as ossein) of the organic substances of the bony structure. It also occurs in the cartilaginous tissues as chief constituent, but it is here mixed with otlier substances, producing what was formerly called chondrigen. Collagen from different tisues has not f{uite the same composition, and probably there are several varieties of collagen.
By continuously boiling with water (more easily in the presence of a little acid) collagen is converted into gelatin. IIofmeister ' found that gelatin, on being heated to 130" C, is again transformed into collagen; and this last may be considered as the anhydride of gelatin. Collagen and gelatin have about the same composition :'
C H X S-fO
Collai,^eii 50.75 • 6.47 17.86 24.92 (Hofmeister)
GoliUiu (from hartshorn) 49.31 6.55 18.37 25.77 (Mulder)
Gelatin (from boues) 50.00 6.50 17.50 26.00 (Premy)
Purilieil gelatin 50.14 6.69 18.12 (Paal)
Gelatin contains regularly small amounts of sulphur which probably belongs to the gelatins and does not exist there as an impurity from the proteids. Vax Name ' has obtained a gelatin from connective tissue, which had been digested with an alkaline pancreas extract (2.5 p. m. Na^COJ for five days, which contained on an average 0.256,'^ sulphur. C. Mokxeh* has prepared a typical gelatin, with only 0.2^ sulphur, by extracting com- mercial gelatin for several days with 1-5 p. m. caustic potash.
Tiie decomposition products of collagen are the same as those of gelatin. Gelatin under similar conditions as the proteids yields amido-acids, sucli as lencin, aspartic and glutamic acids, but no tyrosin, which is especially important. It yields, on the contrary, large qitan titles of glycocoll, to which the name gelatin sugar is given on account of its sweet taste. Lysin and lysatin have also been obtained from gelatin by Drecrsel and E. Fischer, and arginin by IIedijt. ' On putrefaction gelatin yields neither tyrosin, indol, nor skatol," in which it differs from the proteids. Still the aromatic group is not absent in gelatin, and it acts like the oxidized proteid, the oxyprotsulphonic acid, because it yields benzoic acid (Maly ').
' Zeitschr. f. physiol. Chem., Bd. 2.
' Hofniei.«ter, 1. c. ; Mulder, Annal. d. Chem. u. Pharm., Bd. 45- Fremy, Jahresber. d. Chem.. 1854; Paal, Ber. d. deutsch. chem. Gesellsch., Bd. 25. ^ Jouru. of Exp. Med., Vol. 2. •• Private communication from Miirner.
* See Drechsel, Der Abbau der Eiweisskorper. Du Bois-Reymond's Archiv, 1891 ;— Hedin, Zeitschr. f. physiol, Chem., Bd. 21.
* See literature on the cleavage products of gelatin : Drechsel in Ladeaburg's Hand- wOrterbuch, Bd. 3.
' Monatshefte f. Chem., Bd. 10.
56 THE PROTEIN SUBSTANCES.
Collagen is insolnble in water, salt solutions, dilute acids, and alkalies, but it swells up in dilute acids. By continuons boiling with water it is converted into gelatin. It is dissolved by the gastric juice and also by the pancreatic jnice (trypsin sohition) when it has previously been treated with acid or heated with water above + '^0° C By the action of ferrous sulphate, corrosive sublimate, or tannic acid, collagen shrinks greatly. Collagen treated by these bodies does not putrefy, and tannic acid is there- fore of great importance in the preparation of leather.
Gelatin or glutin is colorless, amorphous, and transparent in thin layers. It swells in cold water without dissolving. It dissolves in warm water, forming a sticky liquid, which solidifies on cooling when sufficiently con- centrated. The quantity of ash contained in gelatin is of the greatest importance in the gelatinization of gelatin solutions, as shown by 0. Nasse and A. Kkuger,"'' namely, a diminished quantity of ash diminishes the gelatinizing power.
Gelatin solutions are not precipitated on boiling, neither by mineral acids, acetic acid, alum, lead acetate, nor mineral salts in general. A gelatin solution acidified with acetic acid may be precipitated by potassium ferrocyanide on carefully adding the reagent. Gelatin solutions are precipi- tated by tannic acid in the presence of salt; by acetic acid and common salt in substance; mercuric chloride in the presence of HCl and JSTaCl; metaphosphoric acid, phosphomolybdic acid in the presence of acid; and lastly by alcohol, especially when neutral salts are present. Gelatin solu- tions do not diffuse. Gelatin gives the biuret reaction, but not Adamkie- "Wicz's. It gives Millon's reaction and the xanthoproteic acid reaction so faintly that it probably occurs from an impurity consisting of proteids. According to Morxer, pure gelatin gives a beautiful Milloist's reaction, if not too much reagent is added. In the other case no reaction or only a faint one is obtained.
By continuous boiling with water gelatin is converted into a non-gelatin- izing modification called /S-glutin by Nasse. According to Nasse and Kruger the specific rotatory power is hereby reduced from — 167.5° to about — 136°.' On prolonged boiling with water, especially in the presence of dilute acids, also in the gastric or tryptic digestion, the gelatin is trans- formed into gelatin albumoses, so-called gelatoses and gelatin ])epiones., which diffuse more or less readily.
According to IIofmeister two new substances, semigluiin and liemi- collin, are formed. The former is insolnble in alcohol of 70-80,*^ and is precipitated by platinum chloride. The latter, whicli is not precipitated
' Killinc find Ewald, Verb. d. imliirhist. med. Vcrciiis in Heidelberg, 1877, Hd. 1.
» See Maly's Jahrcsber., Bd. 19.
* In regard to the rotation of /J-glutin, see Franini, PUUger's Arch., Bd. G8.
RETICULIN. 67
by platinum chloride, is soluble in alcohol. Chittenden and Solley ' have obtained in the peptic and tryptic digestion a proto- and a deutero- gelatose, besides some true peptone. The elementary composition of the gelatosea does not essentially dilTer from that of the gelatin. On compara- tive analyses of gelatin, deuterogelatose and gelatin peptone, Chittenden* and his pupils tind nearly the same elementary composition for the gelatin and gelatose, while the gelatin peptone was about 2^ poorer in carbon and about 0.6,<^ poorer in nitrogen than the gelatin. Paal' has prepared gelatin peptone hydrochlorides from gelatin by the action of dilute hydro- chloric acid. Some of these salts are soluble in ethyl and methyl alcohol, and others insoluble therein. Tiie peptones obtained from these salts contain less carbon and more hydrogen than the glutin from which they originated, showing that hydration has taken place. Tiie molecular weight of the gelatin peptone as determined by Paal by IiAOULt's method was 200 to 352, while that for gelatin was 878 to 960.
Collagen may be obtained from bones by extracting them with hydro- chloric acid (which dissolves the earthy phosphates) and then carefully removing the acid witli water. It may be obtained from tendons by extracting with lime-water or dilute alkali (which dissolve the proteids and mucin) and then thoroughly washing with water. Gelatin is obtained by boiling collagen with Avater. The finest commercial gelatin always contains a little proteid, which may be removed by allowing the finely divided gelatin to swell up in water and thoroughly extracting with large quantities of fresh water. Then dissolve in warm water and precipitate with alcohol.
Collagen may also be purified from proteids as suggested by Van Xame by digesting with an alkaline trypsin solution or by extracting the gelatin for days with 1-5 p. m. caustic potash, as suggested by Mokner. The typical properties of gelatin are not changed by this.
Chondrin or cartilage gelatin is only a mixture of glutin with the specific constituents of the cartilage and tlieir transformation products.
Reticulin. The reticular tissues of the lymphatic glands contain a variety of fibres which have also been found by Mall in the spleen, intestinal mucosa, liver, kidneys, and lungs. These fibres consist of a special substance, reticulin, investigated by Siegfried.*
Reticulin has the following composition: C 52.88; 11 6.97; N 15.63; S 1.88; P 0.3-4; ash 2.27. The phosphorus occurs in organic combination. It yields no tyrosin on cleavage with hydrochloric acid. It yields, on the contrar}', sulphuretted hydrogen, ammonia, lysin, lysatinin, and amido-
' Hofmeister, Zeitschr. f. pbysiol. Cbem., Bd. 3; Chittenden and Solley, Journ. of Physiol., Vol. 12.
* Amer. Journ. of Physiol., Vol. 2.
* Ber. d. deutsch. chem. Gesellsch., Bd. 2.3.
•• Mall, Abhandl. d. math. phys. Klasse d. Kgl. sachs. Gesellsch. d. "VViss., 1891. Siegfried, Ueber die chem. eigenscli. der rcticulirten Gewebe. Habil-Schrift. Leipzig, 1892.
58
THE PROTEIN SUBSTANCES.
valerianic acid. On continnons boiling with water, or more readily witli dilate alkalies, reticnlin is converted into a body which is precipitated by acetic acid, and at the same time phosphorus is split off.
Eeticulin is insoluble in water, alcohol, ether, lime-water, sodium carbonate, and dilute mineral acids. It is dissolved, after several weeks, on standing with caustic soda at the ordinary temperature. Pepsin hydro- chloric acid or trypsin do not dissolve it. Eeticulin responds to the biuret, xanthoproteic, and Adamkiewicz's reactions, but not with Millon's
reagent.
It may be prepared as follows, according to Siegfried: Digest intes- tinal mucosa with trypsin and alkali. Wash the residue, extract with ether, and digest again with trypsin and then treat with alcohol and ether. On careful boiling with water the collagen present either as contamination or as a combination with reticulin is removed. The thoroughly dried residue consists of reticulin.
Ichthylepidin is an organic substance, so called by Morner.^ which occurs with col- lagen in tisbscaks and form about i of the organic substance of the same. This substance with 15.9^ nitrogen and 1.1,^ sulphur stands on account of its properties rather close to elastiu. It is insoluble in cold and hot water, as well as in dilute acids and alka- lies at the ordinary temperature. On boiling with these it dissolves. Pepsin hydro- chlor/c acid, as well as an alkaline trypsin solution, also dissolve it. It gives beautiful reac^ODS with Millon's reagent, xanthoproteic reaction, and the biuret test. At least a part of the sulphur is split ofE by the action of alkali.
Skeletins are a number of nitrogenized substances which form the
skeletal tissue of various classes of invertebrates so designated by Keukeis"-
BERG." These substances are cJiitin, spotigin, conchiolin, cornein, and
fibroin (silk). Of these chitin does not belong to the proteinsubstances,
and fibroin (silk) is hardly to be classed as a skeletin. Only those so-called
skeletins will be given that actually belong to the protein group.
Spongin forms the chief mass of the ordinary sponge. It gives no gelatin. On boil- ing with acids, according to the older statements it yields leucin and glycocoll and no tyrosin. Zalocostas claims to have found tyrosin and also butalanin and glycalanin (C5H12N2O4). After HuNDESiiAGEN had shown the occurrence of iodine and bromine in organic combination in different sponges and designated the albumoid containing iodine, iodospongia, Hamack^ later isolated from the ordinary sponge, by cleavage with mineral acids, an iodospongin which contained about % iodine and 4.5^ sulphur. Conchiolin is found in the shells of mussels and snails and also in the egg-shells of these animals. It yields leucin but no tyrosin. The Byssus contains a substance, closely related to conchiolin. which is soluble with difficulty. Cornein forms the axial system of the Autipalhes and Gorgonia. It gives leucin and a crystallizable substance, cornicrys- talliii. According to DuECnsEL ■* the axial system of the gorgonia cavolini contain nearly S'/o of the dry substance in iodine. The iodine occurs in organic combination with n iodized albumoid, gorgonin, Avhich is a cornein. Drechsel obtained leucin, tyrosin, lysin, ammonia, and an iodized amido acid, iodogorgonic acid, which has the composition of a monoiodo-amido butyric acid, as cleavage products of gorgouin. Fibroin and Sericin are the two chief constituents of raw silk. Qy the action of superheated water the sericin dissolves and gelatinizes on cooling (silk gelatin), while the more difficultly soluble fibroin
' Zeitschr. f. physiol. Chem., Bd. 24.
* GrundzUge einer vergl. Physiol, d. thier. Geriistsubst. Heidelberg, 1885.
» Zalocostas, Compt. rend., Tome 107 ; Hundeshagen, Maly's Jahresber., 1895 ; Har- nack, Zeitschr. f. physiol. Chem., Bd. 24.
* Zeitschr. f. Biologie, Bd. 33.
PROTAMINS. 59
reTnains undissolved in the shape of the original fibre. On boiling witii acid tlie fibroin yieids alaiiin (Weyl'), glycoeoli, and a great deal of lyrosin. Fibroin is dissolved in odd concentraled liyilrochloric iii:id with Ibf explusion of l,"? nitrogen as ammonia, aiid it is converted into another, nearly related substance called sn-icoin (Weyl). Sericin yields no glycocoil, but leucin and serin (amidoethylenlactic acid). Tlie composition of the jibove-meutioued Uodies is as follows):'
C H N S O
Conchiolin (from snail-eggs) 50 92 6.88 17.86 0.31 24.34 (Kuukenberg)
Spongin 46.50 6.30 16 20 0.5 27.50 (Ckoockewitt)
48.75 6.35 16.40 (Pohkelt)
Cornein 48.96 5.90 16.81 .... 28.33 (Kuukenbekg)
Fibroin 48.23 6.27 18.31 .... 27.19 (Cka.meh)
'• 48.30 6.50 19.20 .... 26.00 (Vignon)
Sericin 44.32 6.18 18.30 .... 30.20 (Cuamer)
Appendix to Chapter II.
A. PROTAMINS AND HISTONS.
Protamins. In close relationship to the proteids stands a group of sub- stances, the protamins, discovered by Miescher, which are designated by KossEL as the simplest proteids or as the nucleus of the protein bodies. They correspond to the proteids in that they give the three basic bodies, lysin, arginin, and histidiu, on cleavage but differ from the proteids, amongst other things, in not yielding any amido-acids as cleavage products. RUPPEL ' has found that the watery extract of finely divided tubercle bacilli when faintly alkaline or completely neutral has the property of precipitating certain proteids from their solution. This property is dependent upon a substance precipitable by acetic acid which he considers as a combination of a protamin tuberculosamin with a nucleic acid, tuberculinic acid. Free nucleic acid exists in the watery extract, although the reaction is faintly alkaline or neutral (?).
Protamin was discovered by Miescuer* in salmon spermatozoa. Later KossEL isolated and studied similar bases from the spermatozoa of herring and sturgeon. As all these bases are not identical, Kossel uses the name protamins to designate the group and calls the individual protamins sahnin, chipein, and sturin. Kurajeff " has prepared a protamin from
' Ber. d. deutsch. chem. Gesellsch., Bd. 21.
* Krukenberg, Ber. d. deutsch. chem. Gesellsch., .Bdd. 17, 18, and Zeitschr. f. Biologic, Bd. 22 ; Croockewitt, Annal. d. Chem. u. Pharm., Bd. 48 ; Posselt, ibid., Bd. 45; Cramer, Journ. f. prakt. Chem., Bd. 96 ; Viguon, Compt. rend., 115.
3 Zeitschr. f. physiol. Chem., Bd. 26.
♦ In regard to protamins, see Miescher in the histo-chemical and physiological works of Fr. Miescher, Leipzig, 1897; Piccard, Ber. d. deutsch. chem. Gesellsch., Bd. 7; Schmiedeberg. Arch. f. e.xp. Path. u. Pharm., Bd. 37; Kossel, Zeitschr. f. physiol. Chem., Bd. 22 (Ueber die basischeu Stoffe des Zellkerns) and Bd. 25, S. 165 and 190, and Sitzungsber. der Gesellsch. zur Beford. der ges. Naturwiss. zu Marburg, 1897 ; Kossel and Mathews, Zeitschr. f. physiol. Chem., Bdd. 23 and 25.
'Zeitschr. f. physiol. Chem., Bd. 26.
60 THE PROTEIN SUBSTANCES.
the spermatozoa of mackerel, which he calls scombrin, which stands close to clnpein (or salmin), but is not identical therewith. The simplest formula for the snlphate is C,„H.„N,,0,2H,S0,.
The protamins are substances rich in nitrogen (30^ N or more) of a basic nature. Salmin, which is identical with clupein (Kossel), has the formula C,jH,gN,02, according to Mieschee and Schmiedeberg, and CjJI^lSr^O, , according to Kossel. Sturin has probably the formula CjjHjgNjgO,. These statements of Kossel as to the composition of clupein (or salmin) have been found incorrect by recent investigations of the same author. On heating with dilute mineral acids, as also by tryptic digestion, the protamins first yield protamin peptone, protone, from which the three bases, lysin, arginin, and histidin, are derived on further cleavage (Kossel and Mathews). A molecule of salmin, according to Kossel, yields a molecule each of histidin and lysin besides three molecules of arginin. Sturin, en the contrary, yields one molecule histidin besides three molecules arginin and tAVO molecules lysin. Neither lysin nor histidin, but only arginin, occurs in clnpein, which is also true for scombrin. The other con- stituents of the molecule of these protamins are still unknown. Kossel washable to detect a body with the composition of amido-valerianic acid in clupein. We must wait for further elucidation as to the nature of the protamius before we can give anything positive as to the relationship of these bodies to the protein substances.
Solutions of these bases in water are alkaline and have the property of giving precipitates with ammoniacal solutions of proteids or primary albumoses. These precipitates are called histons by Kossel. The salts with mineral acids are soluble in water, but insoluble in alcohol and ether. They are more or less readily precipitated by neutral salts (NaCl). Among the salts of the protamins the sulphate, picrate, and the double platinum chloride are the most important and are used in the preparation of the protamins. The protamins are like the proteids, lasvogyrate. They give the biuret test beautifully, but not Millon's reaction. The protamin salts are precipitated in neutral or even faintly alkaline solutions by phospho- tungstic acid, tungstic acid, picric acid, chromic acid, and alkali ferro- cyanides. Tlie two protamins salmin (clupein) and sturin differ from each other chiefly by a different composition, different solubilities, and somewhat different behavior of the sulphate.
The protamins are prepared, according to Kossel, by extracting the heads of the spermatozoa, which have previously been extracted with alcohol and ether, with dilute sulphuric acid (1-2^), filtering, and precipitat- ing with 4 vols, of alcohol. The sulphate may be purified by repeated solution in water and precipitation with alcohol, and if necessary conversion into the picrate. Miesciter extracts with very dilute hydrochloric acid, neutralizes the excess of acid, and precipitates the base as the double platinum salt.
msTON. 61
As above remarked, Kossel considers the protamins as tlie simplest proteids. If, as is thus far generally the case, we only consider such bodies true protein substances which on decomposition not only yield basic bodies but also, and chielly, monamido-acids, we are rather inclined to consider, with Kossel, the protamins as the nucleus of the proteids, so as not to entirely destroy our conception of protein bodies. Still, before we admit this, the two following conditions must be elncidated: 1. It must be shown that all protein substances yield the three protamin bases as cleavage products, a fact which has not been quite positively confirmed (see Elastin). While IIedix and Bergii ' could not find either lysin, arginin, or histidin among the cleavage products of elastin, still, on the contrary, Kossel and Kutschek' have been able to detect a very small amount of arginin, 0.3^, in the cleavage products of this albumoid. In fibroin, G. Wetzel ^ could either not detect any or only very inconsiderable quantities of basic nitrogen, O.Oftf of the total nitrogen. Conchiolin yielded 8.60,^ of the total nitrogen as basic nitrogen. Among the decomposition products Wetzel found a substance whose hydrochloride showed the same crystallization as histidin hydrochloride, but had a different melting-point. 2. We must obtain further explanation in regard to the molecular weight of peptones, for, as the thing stands at present, the proteid peptone as well as the gelatin peptone, which are generally considered as proteids, have a lower molecular weight (250-400) than the protamins (salmin 751 and sturin 879, according to Kossel).
Histon is the name given by Kossel ^ to a substance isolated by him from the red corpuscles of goose-blood. It is similar in certain behavior to the peptones in the old sense (the albumoses). This histon has the same amount of carbon and hydrogen as ordinary proteid, but contains somewhat more nitrogen, about 18^. When prepared, as suggested by Kossel, from blood-corpuscles by extraction with hydrochloric acid, precipitation of the acid solution by rock salt, and dialyzation until free from salt, it gives the three following characteristic reactions in neutral, salt-free solution: 1. The solution does not coagulate on boiling. 2. With ammonia the salt- free solution gives a precipitate insoluble in an excess of the ammonia. 3. Nitric acid caused a precipitate, which disappeared on warming, and reappeared on cooling.
Later bodies have been described as histous which show a different behavior in one way or another. Liliexfelu has prepared a histon from
» Zeitsclir. f. physiol. Chcm., Bd. 25.
^ Ibid., B(l. 25, S. 551.
*Ibid., B(l. 26.
* Kossel, Zeitscbr. f. physiol. Cliem., Bd. 8, and Sitzungsber. der Gesellsch. zur BefOrd. d. ges. Wissenscb. zu Marburg, 1897 ; Lilienfeld, Zeitscbr. f. pbysiol. Cbem., Bd. 18 ; Scbulz, ibid., Bd. 24 ; Mathews, ibid., Bd. 23.
62 TEE PROTEIN SUBSTANCES.
leucocytes, whose solntion coagalated on boiling, yielding a coagnlnm readily soluble in mineral acids. This histon acted like Kossel's histon ■with ammonia. Sciiulz considers the proteid, globin, set free on the cleavage of haemoglobin, as a histon, although it is extremely soluble in ammonia and does not dissolve in an excess of ammonia, only in the presence of ammonium chloride. Mathews has isolated a body, which he calls arhacin, from the spermatozoa of the sea-urchin (arbacia), and which he considers as a histon, but which differs from the other histons in that it cannot be precipitated by ammonia. The neutral solution of this histon is precipitated by the above-mentioned (page GO) protamiu precipitatants. It has not been shown how the other so-called histons act with these pre- cipitants.
It seems that bodies of various kinds have been described as histons, therefore the author does not feel justified in giving a clear and precise definition of histon. According to Kossel the histons are probably com- binations of protamins and proteid.
R^ Hydrolytic Cleavage Products of the Protein Substances.' 1. Monamido Acids.
Leucin, C.HulS^Oj, or amido-caproic acid, more recently called a-amido^isobutylacetic acid, (CH3),CH.CH,.CH(NHJ.C00H. Leucin is formed not only in the trypsin digestion of proteids, but also from the protein substances by their decomposition on boiling with diluted acids or alkalies, by fusing with alkali hydrates, and by putrefaction. Because of the ease with which leucin and tyrosin are formed in the decomposition of protein substances, it is difficult to positively decide whether these bodies when found in the tissues are constituents of the living body or are only to be considered as decomposition products formed after death. Leucin has been found as a normal constituent of the pancreas and its secretion, in the spleen, thymus, and lymph-glands, in the thyroid gland, in the salivary glands, in the kidneys, brain, and liver. It also occurs in the wool of sheep, in dirt from the skin (inactive epidermis) and between the toes, and its decomposition products have the disagreeable odor of the perspiration of the feet. It is found pathologically in atheromatous cysts, ichthyosis scales, pas, blood, liver, and urine (in diseases of the liver and phosphorus
' As it is not -within the scope of this work, we cannot enter into details in regard to all the cleavage products of the protein substances. These may be found in liandbooks of chemistry. For this reason the most important cleavage products of proteids will be given in the appendix to the protein substances, carnic acid and peptones having already been described. For practical reasons the two amido acids, leucin and tyrosin, will be treated of together, although it would je more theoretically correct to treat the acids of the aliphatic and aromatic series separately.
LEUCIN. 63
poisoning). Leuciu occurs often in invertebrates and also in the plant kingdom. On hydrolytic cleavage various protein substances yield different amounts of lencin. Erlenmeyeh and Schuffer obtained 3G-45,^ from the cervical ligament, Coiix 32^ from casein, and Nexcki 1.6-Z^ from gelatin.'
Leucin has been prepared synthetically by IIufner' from isovaleralde- hyde-ammonia and hydrocyanic acid. This leucin is optically inactive. Inactive leucin may also be prepared, as shown by E. Schulze and BossHARD,' by the cleavage of proteids with baryta at 1G0° C. or on heating ordinary leucin with baryta-water to the same temi)erature. The la^vo- rotatory modification may be formed from the inactive leucin by the action of penicillum glaucum. The leucin obtained in the pancreatic digestion of proteids, as well as in their cleavage with hydrochloric acid, seems always to be the dextro-rotatory variety. Cohn ' has, however, obtained a leucin differing from tiie ordinary leucin in the tryptic digestion of fibrin. HuFXER has prepared an isomer of leucin from monobromcaproic acid and ammonia. It is a question whether there exist natural leucins correspond- ing to normal caproic acid. On oxidation the leucins yield the correspond- ing oxyacids (leuciiiic acids). Leucin is decomposed on heating, evolving carbon dioxide, ammonia, and amylamin. On heating with alkalies, as also in putrefaction, it yields valerianic acid and ammonia.
Leuciu crystallizes when jiure in shining, white, very thin plates, usually forming round knobs or balls, either appearing like hyalin or alternating light or dark concentric layers which consist of radial groups of crystals. Leucin as obtained from the animal fluids and tissues is very easily soluble in water and rather easily in alcohol. Pure leucin is soluble with difficulty; according to certain statements it dissolves in about 29 parts of water at ordinary temperatures or little higher, and according to others in 4G parts. This difference may be due, according to Gmelin,' to the fact that the optically active leucins may be variable mixtures of the dextro- and Isvo- rotatory modifications. The inactive leucin is most insoluble. The specific rotation of the ordinary leucin, dissolved in hydrochloric acid, is («')D = + 17.5.
Leucin is readily soluble in alkalies and acids. It gives crystalline com- pounds with mineral acids. If hydrochloric acid leucin is boiled with
* Erlenraeyer and Scboflfer, cited from Maly, Chem. d. Verdauungssjlfto, in Her- mann's Haudb. d. Physiol., Bd. 5, Theil 3, S. 209 ; Cohn, Zeitschr. f. pbysiol. Chem., Bd. 22 ; Nencki, Jouru. f. prakt. Chem. (N. F.), Bd. 15.
2 Journ. f. prakt. Chem. (N. F.), Bd. 1.
^ See Zeitschr. f. pbysiol. Chem., Bdd. 9 and 10.
* Iloppe-Seyler's Handbucb, 6. Aufl., S. 134, and Cohn, Zeitschr. f. pbysiol. Chem., Bd. 20.
* Zeitschr. f. physiol Chem., Bd. 18.
64 THE PROTEIN SUBSTANCES.
aloohol containing 3-4^ HCl long narrow crystalline prisms of hydro- chloric acid leucinethylester melting at 134° are formed. On slowly heating to 170° C. it melts and sublimes in white, woolly flakes which are similar to sublimed zinc oxide. A marked odor of amylamin is generated at the same time.
The solution of leucin in water is not, as a rule, precipitated by metallic salts. The boiling-hot solution may, however, be precij^itated by a boiling- hot solution of copper acetate, and this is made use of in separating leucin from other substances. If the solution of leucin is boiled with sngar of lead and then ammonia be added to the cooled solution, shining crystalline leaves of lencin-lead oxide separate. Leucin dissolves copper oxyhydrate but does not red nee on boiling.
Leucin is recognized by the appearance of the balls or knobs under the microscope, by its action when heated (sublimation test), and by Scherer's test. This last consists in the lencin yielding a colorless residue when carefully evaporated with nitric acid on platinum-foil, and this residue when warmed with a few drops of caustic soda gives a color varying from a pale fellow to brown (depending on the purity of the leucin), and on further concentrating over the flame it agglomerates into an oily drop which rolls about on the foil.
Tyrosin, CJI,,]SrO„ or jy-oxYPHENYL-AMiDOPROPiONic acid, HO.C^H^.- C3H,(]SrH5).COOH, is derived from most protein substances (not gelatin and reticulin) under the same conditions as leucin, which it habitually accompanies. From genuine proteids such as casein 3-4,^, from horn sub- stance 1-0 fo, from elastin 0.25^, and from fibroin about b<fo have been obtained by Weyl and others.' It is especially found with leucin in large quantities in old cheese {Tvpos), from which it derives its name. Tyrosin has not with certainty been found in perfectly fresh organs. It occurs in the intestine in the digestion of albuminous substances, and it has about the same physiological and pathological importance as leucin.
Tyrosin was prepared by Erlenmeyer and Lipp' from p-amido- phenylalanin by the action of nitrous acid. On fusing with caustic alkali it yields p-oxybenzoic acid, acetic acid, and ammonia. On putrefaction it may yield p-hydrocoumaric acid, oxyphenyl-acetic acid, and j)-cresol.
Tyrosin in a very impure state may be in the form of balls similar to leucin. The purified tyrosin, oti the contrary, appears as colorless, silky, fine needles which are often grouped into tufts or balls. It is soluble with difficulty in water, being dissolved by 2454 parts water at -{- 20° C. and 154 parts boiling water, separating, however, as tufts of needles on cooling. It
> See Maly, 1. c, Bd. 5, Tbeil 2, S. 212 ; R. Cobn, 1. c; Weyl, Ber. d. deutsch. chem. Gesellscli., Bd. 21.
■ Ber. d. deutsch. chem. Gesellsch., Bd. 15.
TTliOSIN. 65
dissolves more easily in the presence of alkalies, ammonia, or a mineral acid. It is difficultly soluble in acetic acid. Crystals of tyrosin separate from an aninioniacal solution on the siiontaneous evaporation of the ammonia. The solution of the tyrosin obtained from protein substances by the action of acids has always a faint laevo-rotatory power. Tyrosin prepared synthetically or by decomposition of proteids by baryta is optically inactive.' Tyrosin is not soluble in alcohol or ether. It is identified by its crystalline form and by the following reactions :
Piria's Test. Tyrosin is dissolved in concentrated sulphuric acid by the aid of heat, by which tyrosin-siilphuric acid is formed; it is allowed to cool, diluted with water, neutralized by BaCO, , and filtered. On the addi- tion of a solution of ferric chloride the filtrate gives a beautiful violet color. This reaction is disturbed by the presence of free mineral acids and by the addition of too much ferric chloride.
IIoFMAXX's Test. If some water is poured on a small quantity of tyrosin in a test-tube and a few drops of Millox's reagent added and then the mixture boiled for some time, the liquid becomes a beautiful red and then yields a red precipitate. Mercuric nitrate may first be added, then, after this has boiled, nitric acid containing some nitrous acid.
Scherer's Test. If tyrosin is carefully evaporated to dryness Avith nitric acid on jjlatinum-foil, a beautiful yellow residue (nitro-tyrosin nitrate) is obtained, which gives a deej) reddish-yellow color with caustic soda. This test is not characteristic, as other bodies give a similar reaction.
Leucin and tyrosin may be prepared in large quantities by boiling albuminous bodies or albuminoids with dilute mineral acids. Ordinarily we boil hoof-shavings {'I 2'>arts) with dilute sulphuric acid (5 jiarts concen- trated acid and 13 parts water) for ^-i liours. After boiling the solution it is diluted with water and neutralized while still warm with milk of lime and then filtered. The calcium sulphate is repeatedly boiled with water, and the several filtrates are united and concentrated. The lime is precij^itated from the concentrated liquid by oxalic acid and the precipitate filtered off, repeatedly boiled with water, all filtrates united and evaporated to crystal- lization. "What first crystallizes consists chiefly of tyrosin with only a little leucin. By concentration a new crystallization may be produced in the mother-lifiuor, which consists of lencin with some tyrosin. To separate leucin and tyrosin from each other their different solubilities in water may be taken advantage of in preparing them on a large scale, but surer and better results are obtained by tiie following method of Hlasiavetz and Habermanx.'' The crystalline mass is boiled with a large quantity of water and enough ammonia to dissolve it. To this boiling-hot solution enough basic lead acetate is added until the precipitate formed is nearly white; now filter, heat the light yellow filtrate to boiling, neutralize with
' See Mauthner, Wien. Sitzungsber., Bd. 85, and E. Scbulze, Zeitschr. f. pbysiol. €bem., Bd. 9.
« Aunal. d. Cbem. u. Phann., Bd. 1G9, S. 160.
CG THE PROTEIN SUBSTANCES.
sulphuric acid, and filter while boiling hot. After cooling, nearly all the tjTOsin is precipitated, while the lencin remains in the solution. The tyrosin may be purified by recrystallizing from boiling water or from ammoniacal water. The above-mentioned mother-liquor rich in lencin is treated with H^S, the filtrate concentrated and boiled with an excess of freshly precipitated copper oxyhydrate. A part of the leucin is precipitated, and the residue remains in the solution and partly crystallizes as a cuprous compound on cooling. The copper is removed from the jorecipitate and solution by means of H,S, the filtrate decolorized when necessary with animal charcoal, strongly concentrated and allowed to crystallize. The leucin obtained from the precipitate is quite pure, while that from the solu- tion is somewhat contaminated.
If one is working with small quantities, the crystals, which consist of a mixture of the two bodies, may be dissolved in water and this solution precijiitated with basic lead acetate. The filtrate is treated with H^S, the new filtrate evaporated to dryness, and the residue treated with warm alcohol, which dissolves the leucin but not the tyrosin. The remaining tyrosin is purified by recrystallization from ammoniacal alcohol. Lencin may be purified by recrystallization from boiling alcohol, or by precipitating it as leucin lead oxide, treating the j^recipitate suspended in water with HjS and evaporating the filtered solution to crystallization. In purifying crude/ leucin KoHMANosr ' prejiares the hydrochloric acid comjDound, and purifies by solution in a little water, and recrystallizes by cooling the solu- tion, and from these he prepares the hydrochloric acid leucinethyl ester.
To detect the presence of leucin and tyrosin in animal fluids or tissues the proteids must first be removed by coagulation with the addition of acetic acid and then precipitated by basic lead acetate. The filtrate is treated with H^S, this filtrate evaporated to a sirup or to dryness, and the two bodies in the resid^^e are separated from each other by boiling alcohol and then purified as above stated.
Glycocol 1, or araido-acetic acid. This acid has not been oblained as a cleavage product of true proteids, but only in the cleavage of gelatin and other albuminoids. As glycocol! is of greater interest as a cleavage product of gl3^cocholic acid and certain other conju- gated acids, it will be treated of iu Chapter VIII.
Alanin, CJIvNO, , or a-amido projMouic acid, CH3.CH(NHs)C00H, has been ob- tained by TVevl^ as a cleavage product of fibroin fiom raw silk. Cystin, occurring occa.sionally in the urine, is considered as a derivative of alanin.
Phenylalanin, or «-phenylamido])ropionic acid, C6H5.C'H2.CH(NH2)COOH, first ob- tained by ScnuLZE and BAKBiEia as a cleavage product of vegetable proteid. The for- mation of this acid in the cleavage of casein with hydrochloric acid and tin chloride is also proliablo according to E. Sciiui.ZE.^
Butalanin, (JjI.iNO,, or o-aniidovalerianic acid, CH2(ISrH2)(CHa)3COOH. This acid ■was lirst detected in tl)e pancreas by v. Gorup-Besanez, ihen b)' Schulze and Barbieri in lupin seeds, also by E. and H. Salkowski in the putrefaction of fibrin, meat and gelatin {II. Salkowski), and by Siegfried among the cleavage products of reticulin, and by Zalocostas* among those of spongin.
Tliis acid forms colorless leaves or starry groups of needles. It melts at 157-158° with decomposition. It is readily soluble in water, dissolves with difficulty in boiling alcohol, but is nearly insoluble in alcohol and ether.
' Ber. d. deutsch. chem. Gesellsch., Bd. 30, S. 1980.
^ Ber. d. deutsch. chem. Gesellsch., Bd. 21.
2 Schulze and Barbieri, ihid., Bd. 16 ; E. Schulze, Zeitschr. f. physiol. Chem., Bd. 9.
* V. Gorup-Besan<z, Annul, d. Chem. u. Pharm., Bd. 98 ; Schulze and Barbieri,
ASPARTIC AND GLUTAMIC ACIDS. 67
Aspartic Acid, C,II,XO^, or amido-succixic acu), CJI,(XlIJ.(COOn).^- This acid is obtained in the trypsin digestion of fibrin and gelatin. It may also be obtained by the decomposition of albuminous bodies or albuminoids with acids. IIla'Siwetz and IIahhu.mann' ' obtained 23.8,^ aspartic acid, although not quite pure, from ovalbumin and 0.3;o from casein. It is very widely diffused in the vegetable kingdom as the amid aspakagixe (amido- succinic-acid amid), Avhich seems to be of the greatest importance in the development and formation of the albuminous bodies in the plants.
Aspartic acid dissolves in "^oG parts water at + 10° C. and in 18.G parts boiling water, and crystallizes on cooling as rhombic prisms. The acid prepared from protein substances is optically active, and is dextrogyrate in a solution strongly acid with nitric acid, and dextrogyrate or la^vogyrate iu a watery solution, dependent upon the temperature.' It forms with cojiper oxide a crystalline combination which is soluble in boiling-hot Avater and nearly insoluble in cold water, and which may be used in the preparation of the pure acid from a mixture with other bodies. In regard to methods of preparation see Hlasiwetz and Habermaxx, and E. Schulze.'
Glutamic Acid, CJl^XO^, or amido-ptrotartaric acid, C3lI^(XHJ. (C'OOH)j. Tliis acid was first found am'ong the cleavage products of vegetable proteids by Ritthausen and Kreusler. Since then Hlasiwetz. and Habermaxx have found it among the cleavage jiroducts of animal, proteids and obtained 29^ glutamic acid from casein. It has also been- prepared by Siegfried from the albuminoid, reticnlin.*
Glutamic acid crystallizes in rhombic tetrahedra or octahedra or iu small leaves. It melts at 135-140° with partial decomposition. It dissolves in 100 parts water at 1G° C. and in 1500 parts 80^ alcohol. It is insoluble in alcohol and ether. The glutamic acid obtained from proteids by boiling with an acid is dextro-rotatory, while that obtained by heating with barium hydrate is optically inactive. It forms a beautifully crystalline combination with hydrochloric acid, which is nearly insoluble in concentrated hydro- chloric acid. This combinatior. is used in the isolation of glutamic acid.. On boiling with copper oxyhydrate a beautiful crystalline copper salt, which
Journ. f. prakt. Chem. (N. F.). Bd. 27 ; E. and H. Salkowski, Ber. d. deutsch. chem. Gesellscb., Bd. 16 ; H. Salkowski, ibid., Bd. 31 ; Siegfried, see foot-note, page 57 ; Za- locoslas, Compt. rend., 107.
' Anual. d. Chem. u. Pliarm., Bdd. 159 u. 169.
' See Landolt, Das optiscbe Dreluuigsvermogen org. Substanzen, Braunsckweig^ 1879, and Cook, Ber. d. deutsch. chem. Gesellscb., Bd. 30.
'Hlasiwetz and Habermunn. Annal. d. Chem. u. Pharm., Bd. 1G9 ; E. Scbuize,. Zeitscbr. f. physiol. Chem., Bd. 9.
•• Rittbaus u and Kreusler, Journ. f. prakt. Chem. (N. F.), Bd. 3; Hlasiwetz and Ilabermann, 1. c, Bd. 159; Siegfried, 1. c., foot-note, page 57.
•68 THE PROTEIN SUBSTANCES.
is soluble with difficulty, is obtained. In regard to the preparation of
glutamic acid see Hlasiwetz and Habermann, and E. Schulze.'
Orloff- makes use of the nickel salts in the separation of the various amide acids. Gh'COCoU and alauin give cr3'stalliue salts, which are soluble with difhculty on boiling with an excess of nickel carbonate. Aspartic acids give a non-crystalline nickel salt which is readily soluble, while leuciu does not give any nickel salt on boiling with nickel carbonate.
2. Basic Bodies.
The most important basic products of hydrolytic cleavage of protein snbst:ances are lysin (lysatin), arginin, and histidin. These are called hexon bases by Kossel.
Lysin, C,H,^X^O,, probably diamido-capeoic acid, CJi„(N"Hj2C00H,
is homologous to ornithin (diami do- valerianic acid ?). Lysin has been
obtained by Drechsel and his pupils not only from different proteids, but
also from several albuminoids on boiling them with acids. It is also formed
in the tryptic but not in the pej)tic digestion of proteids, and also in the
cleavage of protamins (Kossel).' Lysin is readily soluble in water, but
does/not crystallize. It is dextro-rotatory, but becomes optically inactive on
hewing with barium hydrate to 150° C. With hydrochloric acid it gives
two hydrochlorides, and with platinum chloride it gives a chloroplatinate
precipitable by alcohol with the composition CJIj.N'^O^.H^PbClg + C,H^OH.
Lysin gives two silver salts, one of which has the formula Ag]Sr03 -j-
€JI,,N,0,, and the other with the formula AgN03 -f C„H,,N,0,.H^^03
(Hedix). It gives no silver combination insoluble in soda (Kossel).
With benzoylchloride and alkali lysin forms a conjugated acid, lysiiric acid,
CJI,„Xj02(C,H^0),0j (Drechsel), which is homologous with ornithuric
acid, and decomposes into benzoic acid and lysin on being heated with
concentrated hydrochloric acid to 140-150° C." Lysuric acid may be used
in the separation of lysin, first preparing the acid barium salt (C. Will-
DENOW ').
Ornithin, CBHiaNiOj, probably diamido-valerianic acid, C4H7(ISrH2)5COOH. It is formed besides benzoic acid in the cleavage of the conjugated ornithuric acid, discovered by Jaffe, and which is eliminated by birds on feeding benzoic acid. It is also pro- duced with urea in the cleavage of arginin with baryta-water (Schulze and Winter- stein*). Ornithin gives a salt crystallizing in broad colorless leaves, with nitric acid. It gives an odor similar to semen ou warming with caustic soda. On putrefaction
' Hlasiwetz and Habermann, Annal. d. Chem. u. Pharm., Bd. 169 ; E. Schulze, Zcit.schr. f. physiol. Chem., Bd. 9.
'•' Cenlralbl. f. d. med. Wissensch., 1897, S. 642.
^ The works on lysin and lysatin may be found in Drechsel : Der Abbau der EiwcissloUe in Du Bois-Reymond's Arch., 1891, and also Hedin, Zeitschr. f. physiol. Chem.. Bd. 21 ; Kossel, ibid., Bd. 25.
* Drechsel, Ber. d. deutsch. chem. Gesellsch., Bd. 28.
* Zeitschr. f. physiol. Chem., Bd. 25.
* .Taffe, ibid., Bdd. 10 and 11 ; Schulze and Wiutersteiu, ibid., Bd. 30.
BASIC BODIES. 69
Elmnokr' has obtained putrescin, which shows that In ornithi'n an amido group fakes the 0 jiosition.
Diamido acetic Acid, CalloNaOj = CII(NII .)A"OOII, was obtained by DniiciiSEL* HiiKiim' tlie cleavage products on boiling proteids with tin and hydrochloric acid. It iryslallizes in prisms and forms a moubenzoyl combination, which is not very soluble iu water, and nearly insoluble iu alcohol, and which is used iu tJie isolation of the acid.
Lysatin or Lysatinin. The formula of this substance is either 0,n,,X,0, or C'JIjjJS'jO -j- 11,0. In the first case the base is homologOTtS to creatiti, CJI,X,0, , in the other case to creatinin, C\II,X,0, and it is for this reason the body is called lysatin as well as lysatinin. This base is formed under the same conditions as lysin, and according to Hedin it is perhaps only a mixture of lysin and arginin.
The base readily deconi])oses, and on boiling with baryta-water it yields urea. It gives a double silver salt with the formula CJIjjN^Oj.IIXO, + AgNOj , which is soluble in water but insoluble in alcohol-ether, and which is used in the separation and i^urification of the base.
Arginin, CJIj^jS'^O,, was first discovered by Schulze and Steigek in etiolated lupin and pumpkin sprouts. It was later detected by Hedin in the cleavage products of horn substance, gelatin, and several proteid bodies. IIedix obtained the following amounts of arginin from horn substance^ gelatin, conglutin, albumin from egg-yolk, ovalbumin, and casein respec- tively: 2.25; 2.G; 2.75; 2.3; 0.8; 0.8^. Schulze and IxOxgger obtained specially large quantities of arginin, about lOfo, from the proteid of the conifer seeds. Arginin also occurs among the products of trypsin digestion (Kossel and Kutscher).
Arginin is a crystalline substance, which yields urea and apparently also ornithin on boiling with barium hydrate (see above). Several crystalline salts and double salts are known of this base, among which the silver salt is the most important. The silver salt, AgNO, + 0,H,^N^O, + ^11,0, sepa- rates on slow crystallization in beautiful prismatic crystals. It is the least soluble of all the silver salts, and is best suited for the isolation of the base. With silver salt and free alkali or barium hydrate, arginin gives an insoluble silver compound (Kossel).'
Histidin, CgH^XjO, , was first discovered by Kossel as a cleavage product of the protamius (sturin). After this it was found by IIedix ^ among the cleavage products of proteids on boiling them with dilute acid, and by Kutscher among the products of trypsin digestion.
Histidin crystallizes in colorless needles or lamellje. Its watery solution is not precipitated by silver nitrate alone, but on the careful addition of ammonia an amorphous precipitate readily soluble in an excess of ammonia
' Ber. d. deutsch. chem. Gesullsch., Bd. 31.
• Ber. d. silchs. Ges. d. Wissensch., Bd. 44.
•'' Sciiulze and Steiger, Zeitschr. f. physiol. Chem., Bd. 11 ; Uedin, ibid., Bd. 21 ; Schulze (and Rongger , ibid., Bd. 24 ; Kutscher, ibid., Bd. 25; Kossel, ibid.
* Kossel, Sitzungsber. d. kgl. Preuss. Akad. d. Wissensch., Bd. 18, and Zeitschr. f. physiol. Chem., Bd. 25 ; Ilediu, ibid., Bd. 22.
70 THE PROTEIN SUBSTANCES.
is obtained. The hydrochloride crystallizes in beautiful lamellated crystals,' It is optically inactive, dissolves rather readily in water, but is insoluble in alcohol and ether. Histidin acts like arginin with silver salt and alkali. Histidin carbonate is precijjitated by mercuric chloride (Kossel).
The principle of the preparation of these bases consists in first precipitat- ing all the bases with phosiDho-tungstic acid, which leaves the amido acids in solution. The precipitate is decomposed in boiling water with barium Jiydroxide and the bases obtained from the filtrate as silver combinations. In regard to details we refer the reader to the above-cited works of Deechsel and Hedin. Kossel first separates the histidin from the other bases by precipitation with mercuric chloride, but according to more recent investi- gations Kossel^ finds that the mercuric chloride method cannot be used as a general method of separating arginin from histidin, because one can never be sure whether or not the histidin is not contaminated with arginin. According to Kossel lysin may be readily prepared as a picrate, which is obtained on adding an alcoholic solution of picric acid to a concentrated watery solution of the free base. Arginin may be separated from lysin by precipitating with silver sulphate and barium hydroxide.
' See Bauer, Zeitschr. f. physiol. Chem., Bd. 22. J « Zeitschr. f. physiol. Chem., Bd. 26.
CHAPTER III. THE CARBOHYDRATES.
We designate with this name bodies which are especially abundant in the jilant kingdom. As the protein bodies form the cliief portion of the solids in animal tissnes, so the carbohydrates form the chief portion of the dry substance of the plant structure. They occur in the animal kingdom only in proportionately small quantities either free or in combinations with more complex molecules, forming compound proteids. Carbohydrates are of extraordinarily great importance as food for both man and animnls.
The carbohydrates contain carbon, hydrogen, and oxygen. The last two elements occur in the same proportion as they do in water, namely, 2:1, and this is the reason why the name carbohydrates has been given to them. This name is not quite pertinent, if strictly considered; because even though we have bodies, such as acetic acid and lactic, which are not carbo- hydrates and still have their oxygen and hydrogen in the relationship to form water, nevertheless we also have a sugar (rhamnose, CgII,,Oj) which has these two elements in another proportion. Heretofore it was thought possible to characterize as carbohydrates those bodies which contained 6 atoms of carbon, or a multiple, in the molecule, but this is not considered valid at the present time. We have true carbohydrates containing less than 6 and also those containing 7, 8, and 9 carbon atoms in the molecule. The carbohydrates have no properties or characteristics in general which differ- entiate them from other bodies; on the contrary, the various carbohydrates are in many cases very different in their external properties. Under these circumstances it is very difficult to give a positive definition of carbo- hydrates.
From a chemical standpoint we can say that all carbohydrates are aldehyde or ketone derivatives of polyhydric alcohols. The simplest carbo- hydrates, the simple sugars or monosaccharides, are either aldehyde or ketone derivatives of these alcohols, antl the more complex carboliydratea seem to be derived from these by the formation of anhydrides. It is a fact that the more complex carbohydrates yield two or even more molecules of the simple sugars when made to undergo hydrolytic splitting.
The carbohydrates are generally divided into three chief groups, namely» monosaccharides, disaccharides, and polysaccharides.
71
72 THE CARBOHYDRATES.
Our knowledge of the carbohydrates and their strnctaral relationships has been very mncli extended by the pioneering investigations of Kiliais'i ' and especially those of E. Fischer.'
As the carbohydrates occur chiefly in the plant kingdom it is naturally not the place here to give a complete discussion of the numerous carbo- hydrates known up to the present time. According to the plan of this work it is only possible to give a short review of those carbohydrates which occur in the animal kingdom or are of special importance as food for man and animals.
Moiiosaccliarid.es.
All varieties of sugars, the monosaccharides as well as disaccharides, are characterized by the termination " ose," to which a root is added signifying their origin or other relations. According to the number of carbon atoms, or more correctly oxygen atoms, contained in the molecule the monosaccha- rides are divided into trioses, tetroses, pentoses, hexoses, hepioses, and so on.
All monosaccharides are either aldehydes or ketones of polyhydric alcohols. The first are termed aldoses and the other ketoses. Ordinary glucosfe is an aldose, while ordinary fruit-sugar (levulose) is a ketose. The difference may be shown by the structural formula of these two varieties of sugar :
Glucose = CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO; Levulose = CH,(OH).CH(OH).CH(OH).CH(OH).CO.OH,(OH).
A difference is also observed on oxidation. The aldoses can be con- verted into oxyacids having the same quantity of carbon, while the ketoses yield acids having less carbon. On mild oxidation the aldoses yield mono- basic oxyacids and dibasic acids on more energetic oxidation. Thus ordinary glucose yields gluconic acid in the first case and saccharic acid in the second.
Gluconic acid = CH,(OII).[CH(Ori)],.COOII ; Saccharic acid = COOH.[CII(OH)],.COOH.
The monobasic oxyacids are of the greatest importance in the artificial formation of the monosaccharides. These acids, as lactones, can be con- verted into their respective aldehydes (corresponding to the sugars) by the action of nascent hydrogen. On the other hand they may be transformed into stereo-isomeric acids on heating with chinolin, pyridin, etc., and the stereo-isomeric sugars may be obtaihed from these by reduction.
' Ber. (1. deutsdi. chem. Gesellscb., Bdd. 18, 19, and 20.
' See E. Fisclier's lecture : " Syutlieseii in der Zuckergrnppe," Ber. d. deutsch. chem. Gesellscb., Bd. 2'd, S. 2114. . An excellent work on Carbohydrates is Tollen's "Kurzes Haudbuch der Kohlebydrate," Breslau, Bd. 2, 1895, and Bd. 1, 2 Auflage, 1898, which gives a complete review of the literature.
MONOSACCITAIilDES. 7a
Nnmerona isomers occur among tlie monosaccliarides, and especially ia the liexose group. Iti certain cases, as for instance in glucose and levulose, we are dealing with a different constitution (aldoses and ketoses), l)ut in most cases we have stereo-iaomerism due to the presence of asymmetric carbon atoms.
The monosaccharides are converted into the corresponding alcohois by nascent hydrogen. Thus akabinose, which is a pentose, CJI,/J^, is transformed into the pentatomic alcohol, arabit, CJI^^O^. The three hexoses, glucose, levulose, and galactose, C,II„0,, are transformed into the corresponding three hexites, sorbite, mannite, and ijulcite, CJI,,0,. In these reductions a second isomeric alcohol is also obtained as in the reduction of levulose besides mannite also sorbite. Inversely, the corresponding sugars may be prepared from polyhydric alcohols by careful oxidation.
Similar to the ordinary aldehydes and ketones the sugars may be made to take up hydrocyanic acid. Cyanhydrines are thus formed. These addition products are of special interest in that they make the artificial preparation possible of sugars rich in carbon from sugars poor in carbon.
As example, if we start from clucose we obtain glucocyanbydrin on the addition of liy- drocyanicacid: CH3(OH).[CH(OH)]^.COH-t-HCN = CH2(OH).[CH(OH)],.CH(OH).CN. Ou the saponiticatioii of glucocyanhydrin tlie corresponding oxyacid is formed: CHalOH) [CH(OM)],.CH(OH).CN + 2H,0 = CH,(OH).[ClI|OH)]4.CHiOH)COOH4-NH3. Ej the action of uasceul hydrogen on the lactone of this acid we obtain glucohentose. CHmO,.
The monosaccharides give the corresponding oximes with hydroxylamin ; thus glucose yields glucosoxime, CH,(OH).[CH(OH)],.CH : N.OH. These combinations are of importance on account of the fact, as found by WoHL,' that they are the starting-point in the building up of varieties of sugars, namely, the preparation of sugars poor in carbon from those rich in carbon.
The monosaccharides are strong reducing bodies, similar to the alde- hydes. They reduce metallic silver from ammoniacal silver solutions, and also several metallic oxides, such as copper, bismuth, and mercury oxides, on warming their alkaline solutions. This property is of the greatest importance in their detection and quantitative estimation.
The behavior of the sugars to phenylhydrazin acetate is of special importance. Their watery solutions first yield hydrazoxes with phenyl- hydrazin acetate, and then osazones on lengthy warming in the water- bath. The reaction takes place as follows :
(a) CH,(OH).[CH(OH)],.CH(OH).CHO + HoKNH.CHi
= CH,(0H).[CH(0H)]3.CH(0H)CH : N.NH.CrH» + H...0. Pheuylglucoshydrazon
• Ber. d. deutsch. chem. Gesellsch., Bd. 2G, S. 730.
74 THE CARBOHTBRATBS.
Vb) CH5(OH)rCH(OH)]3.CH(OH).CH : N.NH.CeHs + H.N.NH.CeHs " = CiI,(0H).[CH(0H)]3.C . CH : N.NH.CeHs
N.NH.CbHs + H=0 + H.. Phenylglucosazon
The hydrogea is not evolved, but acts on a second molecule of phenylhydrazin and splits it into anilin and ammonia :
HoN.NH.CeHs + H, = H.N.CeHs + NH3.
The osazones are yellow crystalline combinations, which differ from -each other in melting-point, sol ability, and optical properties, and hence have received great importance in the characterization of certain sugars. They have also become of extraordinarily great importance in the study of the carbohydrates for other reasons. Thus they are a very good means of precipitating sugars from solution in which they occur mixed with other bodies, and they are of the greatest importance in the artificial preparation -of sugars.
On cleavage, by the short action of gentle heat and fuming hydrochloric acid, the osazones yield phenylhydrazin hydrochloride and so-called osones, bodies which are ketoaldehydes :
^ CH,(OH).[CH(OH)],.C.CH: KNH.C.H,
N.NH.C.H, + 2H,0 -f 3HC1 = 2C,H,.NH.NH,.HC1 + CH,(0H).[CH(0H)]3.C0.CH0.
Osone
The ketoses are obtained from the osones by reduction with zinc dust ^nd acetic acid:
<:!H,(OH).[CH(OH)].CO.CHO + 211
= CH,(OII).[CH(OH)],.CO.CH,(OH).
If we start with an aldose, we do not get the same sugar back again, but an isomere ketose, and in this way we can convert glucose into levulose.
TVe can also pass from the osazones to the corresponding sugars (ketoses) in other ways, namely, by direct reduction of the osazones with acetic acid and zinc dust. The corresponding osamin is first formed, and then on treating with nitrous acid a ketose is obtained:
CH,(OH).[CH(OH)],.C.CII : N.NH.C.H.
N.NII.C.H, + 11,0 + 4H =
Phenylglucosazon
CH,(0II).[CII(0II)]3.C0.CH.(NHJ + C.1I..NH.NII, + C.H^.NH,
Isoglucosaniia
<JH,(0n).[CH(0II)],.C0.CH,(Nnj + UNO,
= cH,(on).[cii(on)]3.co.cn,(OH) + n, + ii,o.
Levulose
MONOSACCUAUIDES. 75
From what has been stated we see that there are various ways of prepar- ing sugars artificially. They may be prepared (1) by the careful oxidation of tlie polyhyJric alcohols; (2) reduction of tlie corresponding monobasic oxyacids; (;3) splitting of the osazone with hydrochloric acid and a reduction of the osone; (4) direct reduction of the osazone and treating the osamin with nitrous acid; (5) syntheses from combinations poor in carbon (see syntheses of the hexoses).
The isogliicosMuiin prepared in the above manner from plienyljjhicosazon is isomeric ■witli another iilncosainin, whicli may be obtained by the cleavage of chitin (see Chapter XVI) with hydrochloric acid. Both glucosanuns give crystalline sails and have re- ducing i.ctions. The glucosamin (from chitin) gives a dexlro-rotator}', non-fermentable sugar Willi nitrons sicid, while isoglucosamin gives Icvnlose. E. Fisciiek is of the opinion that glucosamin is derived fn)m dextrose, and isoglucosauun from levnlose.
Many varieties of sugar form crystalline combinations, which may be considered as osamins, with ammonia, when they are dissolved in ammoniacal methyl alcohol (Lobry DE Bruyn) ' They give no salts with acids, and differ from the other known isomeric osumius in this respect.
As shown by E, Fischer and his pupils ° the aldoses (also pentoses), as well as ketoses, may enter into an ethereal combination with alcohols in the presence of hydrochloric acid. These combinations are called glucostdes. Such glucosideshave not only been obtained with aliphatic alcohols, but also with benzyl alcohol, polyvalent phenols, and oxyacids. The more compli- cated carbohydrates may, according to Fischer, be considered as glucosides of the sugar. Thus maltose is the glucoside and lactose the galactoside of grape-sugar.
By the action of alkalies, even in small amounts, as also of alkaline earths and lead hydroxide, a reciprocal transformation of the sugars, such aa glucose, levulose, and mannose, may take place (Lobry de Bruyn and Alberda VAX Ekensteix).'
Two other sugars, among them two ketoses, are produced by the action of potash or soda on each of the tliree sugars, glucose, levulose, and galactose. For example, from glucose two ketoses, levulose and pseudoleviilose, are produced, also mannose and a non- fermentable sutiar, glutose. From galactose are formed talose and galtose, besides two ketoses, tagatose and pseudotagatose.
The monosaccharides are colorless and odorless bodies, neutral in reac- tion, with a sweet taste, readily soluble in water, generally soluble with difiiculty in absolute alcohol, and insoluble in ether, and some of which crystallize well in the pure state. They are optically active, some lajvo-