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casein, albumin, and other animal proteids. The glutaminic acid they isolated
was levorotatory, while that obtained by Schiitzenberger was only found in
small quantities and was optically inactive. By boiling the active form with
barium hydrate, however, it is converted into the inactive form, and further,
the inactive form if allowed to ferment under the action of the mould
Penicillium glaucum, is transformed into the optically active modification.*'

Employing the same method, Horbaczewski '' obtained leucine, glutaminic acid,
and glycocine from gelatin, but no asparaginic acid or tyrosine. Hofmeister,^
however, obtained small quantities of asparaginic acid.

Prom elastin were obtamed ammonia, leucine, tyrosine, glycocine, butalanine
(CjH;^-^^©.,, ami do-valeric acid), but no glutaminic or asparaginic acids. ^

From reticulin (a substance separated from reticular tissue) were obtained
ammonia, sulphuretted hydrogen, and amido-valerie acid, but no tyrosine or
glutaminic acid (Siegfried).

By the emplojnnent of the same method, Drechsel^^ has discovered two
new substances, which are of special interest. He began by studying
the quantities of nitrogenous bodies which other observers had obtained ;
and if he assumed that only one-half of these had been isolated there
still remained about 30 per cent, of the nitrogen of the proteid to be
accounted for. He pointed out also that Schiitzenberger obtamed
carbonic anhydride by his method, and that Hlasiwetz and Habermann
did not by theirs. He argued from this that there shoidd be some other

^ Xeumeister, Ztschr. f. Biol., Miiucben, Bd. xxiii. S. 381.

- See Brodie, Scieiiee Progress, London, 1895, vol. iv. p. 67.

'^ Ann. d. Chem., Leipzig, 1873, Bd. clxix. S. 150. In a paper published previous to
this by the same ob-servers [ihid., Bd. clix. S. 304), they sought unsuccessfully to establish a
definite relationship between proteids and carbohydrates.

^ Journ. f. praJd. Chem., Leipzig, Bde. xcix. S. 454 ; cvii. S. 218.

^ Silzungsb. d. k. Akad. d. Wissensch., Wien, 1872, Bd. ix. S. 114.

^ Bcr. d. dcutsch. cJiem. Gesellsch., Berlin, Bd. xvii. S. 388. Similar changes in the
optical varieties of leucine and other substances are similarly produced.

^ Sitzungsb. d. k. Akcul. d. Wissensch., Wien, 1880, Abth. 2, Bd. Ixxx. S. 101.

* Ztschr. f. physiol. Cliem., Strassburg, Bd. ii. S. 299.

^ Horbaczews^, Sitzungsb. d. k. Akad. d. Wissensch., AVien, 1886, Abth. 2, Bd. xcii.
S. 657.

" "Der Abbau d. Eiweissstoffe, " Arch. f. Physiol, Leipzig, 1891, S. 248,



THE DECOMPOSITION PRODUCTS OF PROTEIDS 33

substance produced by the latter method, which should yield carbonic
anhydride on treatment with an alkali. As he considered it probable
that this substance might l)e basic, he endeavoured to isolate it as a
precipitate by means of pliosphotungstic acid, a reagent which is of
great value as a precipitant of basic or alkaloidal bodies. By this means
he succeeded in isolating not one but two bases, which he named lysine
and lysatinine.

Lysine has the formula CCH14N2O2, and is homologous with Jaffe's ^
ornithin (C5H12N2O2, proljaljly diamidovalerianic acid). Lysine is pro-
bably diamidocaproic acid. On heating it to 120°-130° with barium
hydrate, barium carbonate is formed. It will therefore account for some
of Schiitzenberger's carbonic anhydride.

Lysatine or lysatinine, the second base, is even more interesting.
Its formula is either C6II13N3O2 or CeHi^NaO, and is a homologue of
either creatine (C4H9N3O2) or creatinine (0411^^30), according as the first
or second formula is taken.

It can be obtained pure as a double salt of its nitrate with silver
nitrate, and this when boiled with barium hydrate for twenty-five
minutes yields urea ; this is a decomposition exactly analogous to that of
creatinine under the same circumstances. From 10 grms. of the
double salt Drechsel obtained about 1 grm. of lu^ea ; which is a large
quantity when one considers that under the conditions urea is quickly
broken up into ammonia and carbonic anhydride.

This is the first time that urea has been obtained from proteid in laboratory
experiments. Many years ago, Bechamp ^ stated that he had obtained urea
from egg-white by the oxidising action of potassium permanganate. Lessen ^
found that the substance Bechamp took for urea was guanidine, which
probably came from small quantities of xanthine, present in the egg-white as
an impurity, or, as Drechsel '^ points out, from lysatinine. Drechsel's method,
it is important to notice, is one of hydration; not, like Bechamp's, one of
oxidation.^

It should be added that Drechsel's work was carried out in the first
instance with casein, but his pupils have discovered lysine and lysatinine
among the cleavage products of other proteids and proteid-like substances,''
and further that the same substances are formed during pancreatic digestion.'^

Hedin ^ has more recently arrived at the conclusion that lysatinine is not
a chemical individual, but a mixture of lysine with another base called
arginine. Arginine (0(511^4^40.2) was originally separated from vegetable
tissues by Schulze and Steiger,^ and subsequently it was found by Hedin ^'^

^ Ber. d. deutscli. chem. Gesellsch., Berlin, Bd. x. S. 1925.

2 Ann. d. Cliem., Leipzig, 1856, Bd. 100, S. 247.

" Ibid., 1880, Bd. 201, S. 369. Between Bcecliamp's and Lossen's time the question was in-
vestigated by Stadeler, Journ. f. prakt. Chem., Lei])zig, 1857, Bd. Ixxii. S. 251 ; Loew, ibid.,
1871, N.F., Bd. iii. S. 289 ; Tappeiner, ibid., 1871, Bd. civ. S. 408 ; and Ritter, Compt.
rend. Acad. d. sc, Paris, tome Ixxiii. ]>. 1219 ; all of whom except Ritter failed to confirm
B(5champ's results.

■* Loc. cit.

^ On the formation of urea by oxidation from many organic substances, see F. Hofmeister,
Arcli. f. exper. Path. u. Pharmakol., Leipzig, 1897, Bd. xxxvii. S. 426.

" Fischer (from gelatin), Inaug. Diss., Leipzig, 1890; Arch. f. Anat. u. Physiol., Leipzig,
1891, S. 205. Siegfried (from conglutin), Ber. d. deutsch. chem. Gesellsch., Berlin, Bd. xxiv.
S. 418 ; (from reticulin), " Ueber d. chem. Eigenschaften des retic. Gewebes," Habilitation-
schrift, Leipzig, 1892. Siegfried also obtained from conglutin a sweet substance
(CgH, cN.^O^), corresponding to one of Schiitzenberger's gluco-proteins.

■^ Hedin, loc. cit. ^ Ztschr. f. jjhysiol. Chem., Strassburg, 1896, Bd. xxi. S. 297.

^ Ibid., Bd. xi. S. 43; Ber. d. deutsch. chem. Gesellsch., Berlin, Bd. xxiv. S. 2707 ; 1396,
Bd. xxix. S. 352.

1" Ztschr. f. physiol. Chem., Strassburg, Bd. xx. S. 186 ; xxi. S. 155 ; xxii. S.191.
VOL. I. — ^



34 CHEMICAL CONSTITUENTS OF BODY AND FOOD.

to be a constant decomposition product of proteids and albuminoids. It
yields urea on treatment of its silver salt Avitli barium hydrate.

Other recently published work on the decomposition of proteids by
hydrochloric acid is by E. Cohn.^ He used concentrated acid, and no
stannous chloride. From 1000 parts of casein he recovered 916"75 of
products ; these consisted of fatty acids, 34*25 ; tyrosine, 35 ; leucine, 321 ; an
oily product containing an acid, C^H^^glSToOg, 180; pyridine derivatives, 3"65;
other substances, including cystin, cystein, thiolactic acid, and Drechsel's bases,
the remainder.

3. Treatment vntli oxidisioig agents. — Treatment of proteids with
nitric acid yields, first, an insoluble yellow substance of uncertain
composition, called xanthoproteic acid; this dissolves gradually, and
finally paraoxybenzoie acid (probably from the oxidation of tyrosine),^
oxalic, fmnaric, and saccharic acids are formed.

Oxidation by manganese dioxide or potassium bichromate, with
sulphuric acid, has yielded — (1) Fatty acids, from formic up to caprylic
(CgHigOo), and their aldehydes ; (2) nitrates of acetic, propionic, valeric,
butyric, and hydrocyanic acids ; (3) benzoic acid and benzoic aldehyde.
Oxidation with chlorine water has yielded, among other products, fmnaric
acid, oxalic acid, and chlorazoL Oxidation with bromine water at high
temperatures in a sealed tube has yielded carbonic anhydride, oxalic
acid, ammonia, bromanil (CgBr^Og), bromoform (CHBrg), monobrom-
benzoic acid, mono- and dibromacetic acids, tribromamidobenzoic acid
(CeHBr3(NH)C00H), asparaginic and malaminic acids, leucine and
leucimide (CgHuNO). No tyrosine was obtained.^^

4. Treatment ly action of heat. — Dry distillation leads to the forma-
tion of a complex oily material, called " Dippel's oil," which contains
a large number of substances ; among these are hydrocarbons of the
fatty series, ammonium salts of fatty acids, nitrates and ketones of the
same series, carbonic anhydride, ammonia, amines of fatty acid radicles,
hydrocarbons and amines of the benzene series, aniline and its
homologues, phenol and its homologues, members of the pyridine
group of bases, namely, pyridine (C5H5N5), picoline (CgH^N), lutidine
(C7H9N), and coUidine (CgH^iN), and lastly, pyrrol.

Some of the substances last mentioned will also be found in our list of the
animal alkaloids (p. 59) ; here we have direct proof that proteid substances
have within them, or are capable of forming by intramolecular rearrangement,
basic bodies of this nature. The pyridine bases have, moreover, been shown
to take a part in the formation of the vegetable alkaloids, piperidine,
cinchonine, etc.

It is extremely probable that proteids contain within their molecule
a radicle of the closed ring series, but even if they do not, there still
remains the possibility that by the action of heat, substances with
open carbon chains may be transformed into those with closed rings.
The following example is selected by Brodie * : —

^ Ztschr. f. ijliysiol. Chem., Strassbiirg, 1896, Bd. xxii. S. 1.53.

^ Baumann, Ber. d. deutsch. chem. Gesdlsch., Berlin, Bd. xii. S. 1453.

^ Hlasiwetz and Habermann, Ann. d. Chem., Leipzig, 1871, Bd. clix. S. 304. Blennard,
Compt. rend. Acad. d. sc, Paris, tome xc. p. 612; xcii. p. 458. This latter observer also
obtained glnco-proteins.

■* Loc. cii.



THE ATTEMPTED SYNTHESIS OF PROTEIDS. 35

At 190° dry glutaminic acid is converted into pyrogiutaminic acid,
and at a higher temperatiu'e this, in turn, is converted into pyrrol.^



COOH

/


COOH


CH:GH

1 \


/
CHo GH.XHo


CH„— CH

1 ' \




\
;.XH


CHo COOH.




;nh


/
CH:CH


(glutaminic acid)


/

CHo— CO

(pyrogiutaminic acid)


(pyrrol)



The attempted synthesis of proteids. — Since Wohler in 1828
succeeded in making lu'ea artificially from its elements, the strides
that organic chemistry has made have been prodigious. Complex
substances, previously made only in the li\4ng laboratory of plants
and animals, are now manufactm^ed daily in the test tubes and retorts
of the chemist. The substances of most importance to vital processes,
the carbohydrates and the proteids, have been among the last to yield
before this advance, but we have seen how sugar has given way to
Fischer ; and there are signs that the last conquest of organic chemistry,
the synthesis of proteids, cannot be far distant. I propose to sketch one
or two of the principal attempts that have been made in the manu-
facture of albuminous from simpler substances.

Schutzenherger s experiments. — We have ah^eady seen that the pro-
ducts of decomposition of a proteid are extremely nimierous, but l;)riefly
they fall into two principal groups, the fatty compounds (generally con-
tainmg an amidogen radicle) and the aromatic compounds or derivatives
of benzene. To Schiitzenberger ^ belongs the credit of an attempt to
build up from some of the compounds he had shown could be obtained
from albumm, something like the original proteid.

In order to effect the synthesis of proteid material, he considered it
necessary to combine a molecule of a leucine {i.e. an amido-fatty acid)
with a molecule of a leuceine (an amido-acid of the acrylic series), with
ehmination of water, and then to combine this complex group with one
or more molecules of lu-ea and oxamide, also with the elimination of
water. We have already seen that the method he had adopted for the
breaking up of proteids — heating with alkali — leads to hydrolysis ; so in
any attempt at synthesis he recognised as a sine qua non the necessity
of some method of dehydration.

The provisional formula he gives is the following : —

with elimination of eight molecides of water. This would give

C.+^Ho^.sNsOs, andifq = 28,

the percentage composition calculated from the formula agrees closely
with that of albumin.



^ Bernheimer, Ber. d. deutsch. chem. Gesellsch., Berliu, Bd. xy. S. 1222.
'^ Comjyt. rend. Acad. d. sc, Paris, tome cvi. p. 1407 ; cxii. p. 198.



36 CHEMICAL CONSTITUENTS OF BODY AND FOOD.

Accordinglj^, amido-compoiinds, leucines (C,„Hoi„+iN02), and leuceines
(CuH.2„_iN0o), were mixed with about 10 per cent, of urea, and finely
powdered. The mixture was dried at 110° C., intimately mixed with 1*5
times its weight of phosphoric anhydride, and heated in an oil bath. At
120° C there is no change; at 125° dehydration takes place rapidly, and
the mixture becomes pasty, but solidifies to a compact solid mass without any
darkening. This is dissolved in water, the solution mixed with excess of
alcohol, and the pasty precipitate so produced washed with alcohol and
re-dissolved in water. Phosphoric acid is removed by baryta, and the filtered
liquid when concentrated yields an amorphous product soluble in water, but
precipitated in a curdy form on the addition of alcohol.

Aqueous solutions of this product are precipitable by most of the other preci-
pitants of proteids ; it gives the biuret and the xanthoproteic reactions. When
burnt it gives the characteristic odour of burning nitrogenous animal matter.

The product he obtained does not give all the proteid reactions;
it is, for instance, not precipitable by acetic acid and ferrocyanide of
potassimn ; and the evidence as to its proteid natui'e is otherwise hardly
conclusive, because the colour tests for proteids are given by many of
the decomposition products of albuminous matter. The partial success
obtained will, however, point the way for futm^e attempts, and so far
as it goes, is in favour of Schiitzenberger's ureide theory of proteid
constitution. Complete success could hardly have been anticipated
from such an experiment, because no means were taken to ensure the
presence of sulphur, an element present in all proteids. Moreover,
in the synthesis, no aromatic substance was introduced; this, how-
ever, is not absolutely necessary, as the formation of aromatic from
fatty compounds by heat is a familiar chemical change (see p. 34).

Grimaux's experiments. — Some years previous to Schiitzenberger's
work, G-rimaux i had obtained, by somewhat simpler processes, substances
which even more resembled proteids in theu^ reactions than Schiitzen-
berger's. He was engaged in studpng the properties of certain sub-
stances, inorganic and organic, which he termed " coUoidcs," and of those
which he prepared the three which especially bear on the present
question are the following : —

(a) Colloide amidobenzoique. — This is made by heating, to 125° C,
meta-amidobenzoic acid in a sealed tube, with one and a half times its
weight of phosphorus pentachloride, for ninety minutes. The product is
a white friable powder ; this is washed repeatedly with boihng water to
remove all phosphoric acid. The remaming substance Grimaux supposes
to be an intramolecular anhydride, formed by the union of several mole-
cules of meta-amidobenzoic acid, with the elimination of water. When
ammonia is added, it dissolves slowly in the cold, but rapidly on heating.
The solution obtamed is evaporated in vacuo, at a low temperature, and
the resulting solid is a transparent jelly which dries into translucent,
yellowish plates, which in their physical properties resemble dried
serum albumin.

(b) A colloid which is similarly prepared, except that the temperature
in the sealed tube is allowed to rise to loh" C.

(c) Colloide aspartiqne. — This is made by the action of a current of
gaseous ammonia on solid aspartic anhydride, heated to 170° C. The

^ Compt. rend. Acad. d. sc, Paris, tome xciii. ]). 771 ; xcvii. pp. 231, 1336, 1434, 1485,
1540, 1578 ; liev. sclent., Paris, April 18, 1886 ; tliis article gives a summary of the other
papers.



THE ATTEMPTED SYNTHESIS OF PROTEWS. 37

product is washed with water, and after evaporation in vacuo yields a
substance similar in appearance to the colloid (a).

In all three cases, heavy molecules were formed ; and in all, the
result was a colloidal substance, exhibiting many of the properties of
proteids. In the case of the first two colloids, there was present not
only the amidogen, but also the aromatic radicle. Although the result
is not albumin, the resemblance between the physical properties and
chemical reactions of proteids and of these synthesised colloids is
remarkably close. Pickering ^ has fully confirmed Grimaux's results, and
has added new facts illustrating the points of similarity between them
and proteids.

The chief of these are as follows : —

1. All give the xanthoproteic reaction.

2. With copper sulphate and caustic potash, a gives a blue-violet ;
h, nil ; c, a typical violet coloration, like that given Ijy albumin.

3. Theu' solutions do not coagulate on heating in the absence of salt ;
if, however, a trace of a soluble barium, strontimn, or calciimi salt is
present, opalescence occurs at 56°, and coagulation at 75° C.

4. The colloids are removed from solution (rising to the surface of
the fluid) by saturation with magnesium sulphate, sodium chloride or
ammonium sulphate. Here they especially resemble the class of
proteids called globulins.

5. Another resemblance to globulins is seen in their behaviour to a
stream of carbonic anhydride, which, in the presence of salts, causes
precipitation. The passage of a current of air through the mixture
redissolves the precipitate.

6. The colloid & is not digested by pepsin-hydrochloric acid ; a is
slightly digested ; c is easily digested, and the solution gives the typical
peptone colour, pink, on the addition of copper sulphate and caustic
potash.

7. Any one of the three colloids, when intravenously injected into
animals (rabbits, cats, dogs, rats, gumea-pigs) causes extensive intra-
vascular coagulation. In a typical experiment death is due to respiratory
failure. In this property of the proteid-like colloids, which was dis-
covered by Pickering, they closely resemble the nucleo-proteids (see
p. 67). The resemblance to the action of the nucleo-proteids extends
even to minor points ; for instance, neither cause intravascular clotting
in albino rabbits ; ^ and in dogs very minute doses indeed, cause a slowing
of the rate of coagulation (Wooldridge's negative phase).

The artificial colloids do not resemble nucleo-proteids in promoting
the formation of fibrin in solutions of fibrmogen.

If nucleo-proteids and these colloids both produce the same effect in
the same way, their physiological activity is probably connected with
the presence of some radicle common to both. The colloidal condi-
tion will not explain the action, since most colloids do not act thus.

'■Pickering, J. W., Journ. Physiol., Cambridge and London, vol. xiv. p. 347; xviii.
p. 54 ; Pickering and Halliburton, ihid., vol. xviii. p. 285. More recently Pickering has
succeeded in making several new proteid-like colloids {Proc. Fmij. Soc. London, 1896,
vol. Ix. p. 337).

^ This point has been worked out by Pickering also in the case of the Arctic hare.
During the albino stage of the animal, neither nucleo-proteids nor synthesised colloids cause
intravascular coagulation, but during their pigmented stage intravascular coagulation is
produced in the usual way. The change in the external appearance of the animal is thus
associated with other changes in its constitution {Journ. Physiol., Cambridge and London,
1896, vol. XX. p. 310).



38 CHEMICAL CONSTITUENTS OF BODY AND FOOD.

The active radicle cannot be one which contains phosphorus, since the
synthesized colloids are free from that element. It may possibly be the
amido-fatty radicle in a high state of condensation.

Lilienfeld and Wolkowicz,^ by the condensation of amido-acid compoimds,
have obtamed substances which resemble proteoses in their reactions.

Theories of proteid. constitution.— The views of Schiitzenberger
on this subject will have been gathered from the preceding section.
There now remain to be mentioned some other theories on the subject,
which are in part deductions from the work of others, and partake
more of the nature of speculation than of hypotheses that have been
tested by experunent.

Pfiugers theory. — The distinction between non-hving proteids and
living protoplasm was noted as early as 1821 by Eudolphi,^ who
wrote : " The components of the dead and living body do not exist under
the same chemical conditions." A few years later the distinction
between living and non-hving proteids was emphasised by John
Fletcher.^ Ptiitger's theory* was, however, the first intelligible one to
explain such differences. The non-living proteids, such as are contained
in white of egg, are stable and indiff'erent to neutral oxygen ; but when
such proteids are assnnilated — that is, liecome part of a living cell — the
molecules live by breathing oxygen. The assimilation of a proteid is
probably due to the formation of ether -hke combinations between
the molecules of livmg proteid and the isomeric molecules of the food
proteid, water being eliminated ; this process of polymerisation produces
large and heavy jjut still shnple molecules ; and during its occurrence
the nitrogen of the non-hving proteid leaves the hydrogen with which it
is combined in the form of amidogen (NHj), and enters into combination
with carbon to form the much more rmstable substance cyanogen (CN).
We thus find uric acid, creatine, guanine, etc., as products of proteid
metabolism, while none of such cyanogen-containmg molecules are
obtainable from non-living proteid.

Pfliiger's theory was put forward in 1876 ; but in the light of Drechsel's
later work, the part involving exchange of nitrogen between cyanogen and
amidogen is rendered unlikely, and with that the whole theory mustprobably fall.

Loevjs theory. — The researches of Loew and Bokorny ^ have taken
the same clu-ection as those of Pfliiger, in that they are attempts to
explain the distinction between livmg and dead protoplasm. Living
protoplasm or proteid (in the cells of various algfe) has the property of
reducmg silver from a weak alkalme solution of silver nitrate ; dead
proteid has no such effect ; animal protoplasm is so quickly killed by
silver nitrate, that it does not give the reaction. The conclusion
formed is, that something of the nature of an aldehyde occurs in living
protoplasm. Formic aldehyde is probably formed in plants by the union
of carbon and water ; if this is inrited to ammonia, aspartic aldehyde is
formed, thus : —

1 Arch./. Anaf. u. Fhysiol., Leipzig, 1894, Pliysiol. Abtb. S. 383 and 555.
^ " Grniidnss der Physiologie," 1821.

* •■'Rudiments of Physiology," Edinburgh, 1837.

* Arch./, d. fjes. Physiol., Bonn, Bd. x."S. 251.

^ " Die cheni. Kraftquelle ini lebenden Protoplasma," Munich, 1882. Loew's most
recent views on this subject will be found in a recently publislied pamphlet, " The Energy
of Living Protoplasm," London, 1896.



GENERAL PROPERTIES AND REACTIONS OF PRO TE IDS. 39
4CHOH+NH3 = NH2.CH.COH+2H,0

CH,.COH

By polymerisation of aspartic aldehyde we have —

rNH.,.CH.coi-n

[ CH.COHJ

and by further polymerisation in the presence of a sulphur compound
and hydrogen we get

which represents the composition of ordinary albumin. If such an
aldehyde does exist in " living proteid " its instability is explicable,
because molecular movements would be constantly occurring in the
aldehyde group.

The theory is ingenious, but an obvious objection to it is that it assumes
the empirical formula above given for albumin to be the correct one. The
theory has been adversely criticised by Baumann ^ who points out that alde-
hydes are not the only substances that reduce alkaline solutions of silver
nitrate, but that many organic substances, such as pyrogallol, resorcin, hydro-
chinon, pyrocatechuic acid, alloxan, and morphine do so also. It is stated,
moreover, by Kretzschmar ^ and Griffiths, ^ that both living and dead proto-
plasm give the reaction.

Lathams theory.^— ThiQ is to some extent a combination of the two
just described. Latham considers living proteid to be composed to a



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