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acids, without passing through the preliminary stages of hemialbumose
and hemipeptone ; at any rate, there is no experimental evidence of such
a passage. Also, when protoalbumose is obtained as a product of
fractional peptic digestion, and submitted to the action of trypsin, it is
directly broken up into amido-acids, no deuteroalbumose or hemipeptone
being discoverable as intermediate products. Similarly, heteroalbumose
is in part converted into amido-acids, and in part into awi^'i-deuteroalbu-
mose, which passes later into antipeptone without any formation of
7i6wi-deuteroalbumose or amphopeptone.^

1 Neumeister, Ztschr. f. Biol., Munchen, 1887, Bd. xxiii. S. 381.



420 CHEMISTR Y OF THE DIGESTIVE PROCESSES.

This is all easily accounted for on the supposition that a variable
fraction of the proteid molecule is easily attacked and broken off into
amido-acids by trypsin, but it is very difficult to explain on the sup-
position that the proteid molecule, early in the process of decomposition,
breaks up into two halves, of which one changes through the stages of
hemialbumose and hemipeptone into amido-acids, while the other,
passing through antialbumose, halts at antipeptone.

Description of the products formed in the pancreatic digestion
of proteids. — The products of tryptic digestion may be isolated most
easily by experimenting with fibrin, either by impregnating it with the
ferment, washing, and allowing it to digest in dilute sodium carbonate
solution, or by digesting with a purified pancreatic extract. The pro-
ducts present at different stages may be studied by removing at intervals
a portion of the digest, stopping the digestive process, by boiling and
then investigating the nature of the dissolved substances.

Goagulcible proteid. — If the test portion be removed before complete sokition,
or just on complete solution of the fibrin, it will be found to contain coagulable •
proteid ; on neutralising, part of this, being a globulin in character, is thrown
out of solution, and the remainder on making faintly acid and boiling.^

Tlie deuteroalbumose of "pancreatic digestion. — If, after removal of the
coagulated proteid by filtration, the solution be now concentrated, deuteroalbu-
mose can be precipitated from it by sodium chloride and acetic acid, and shown,
by subjection to further action of trypsin, to be purely an anti-compound, or,
in other words, to contain nothing in its molecule decomposable by the action
of trypsin into amido-acids. This anti-deuteroalbumose, as already stated, is
the only albumose found in tryptic digestion, and it is only found in the earlier
stages. Another portion of the digest may be acidified, and the albumose
thrown out of solution by saturation with ammoniinn sulphate, after which the
presence of peptone in the filtrate may be shown, after dilution or dialysis, by
the usual tests.

After some days of tryptic digestion, the digest contains no coagulable
proteid or albumose, but only antipeptone, and the simpler products formed
by more complete demolition of part of the proteid molecule (or of the hypo-
thetical hemi-moiety), such as the amido-acids.

The peptone of tryptic digestion or antipeptone. — The peptone or peptones
formed by the action of trypsin on proteids can best be obtained from a
pancreatic digest which has been allowed to proceed to completion by
repeated digestion during several days with trypsin and dilute sodium carbo-
nate solution. This solution is concentrated to a small volume and filtered
from the tyrosine, which separates out on cooling. The filtrate is saturated
with ammonium sulphate, with the precautions described under peptic diges-
tion,^ and the ammonium sulphate is similarly removed. The antipeptone may
now be precipitated by the addition of phosphomolybdic acid, the precipitate
decomposed by baryta water, and excess of barium removed by cautious
addition of dilute sulphmic acid. Finally, the solution is concentrated to a
syrup on a water bath, and dried in vacuo over sulphuric acid.^

Antipeptone agrees very closely in composition and properties with a
monobasic organic acid (Fleischstiure) recently isolated by Siegfried ^ from
muscle extract, of the composition and molecular weight represented by the

1 See pp. 405, 415. " See p. 411.

3 Kiilme, Ztsclir. f. Biol., Munchen, 1893, Bd. xxx. S. 1.

"^ Bfir. d. 1c. sacks. GesdUch. d. JVissensch., Math.-phys. CI., 1893, S. 485; Arch. f.
Anat. n. Physiol., Leipzij,'. 1894, S. 401 ; Ztxchr. f. physiol. Chem., Strassbiirg, 1896, Bd.
xxi. S. 360. See also C. W. Rockwood, Arch. f. Anat. u. Physiol., Leipzig, 1895, S. 1 ;
Bailee u. H. S. Ide, Ztschr. f. 'physiol. Ghcm., Strassburg, 1896, Bd. xxi. S. 380.



AMIDO-ACIDS FORMED IN TRYPTIC DIGESTION. 421

formula C^oH^gNgOj. This substance gives a similar biuret reaction to that
given by antipeptone ; like it also, it does not give Millon's reaction, is very-
hygroscopic, and, on decomposition with hydrochloric acid, forms lysine and
lysatinine, but not tyrosine. It has also been obtained directly from the pro-
ducts of advanced tryptic digestion ; it has been found in milk, and in traces
in the urine. It is easily soluble in water ; sparingly in cold, more so in hot
alcohol, from Avhich it crystallises in microscopic crystals. It is also soluble in
carbolic acid and glacial acetic acid, but is decomposed by these solvents,
especially at a high temperature. It combines with hydrochloric acid and with
phosphoric acid (Phosphorfleischsaure). The compound with phosphoric acid
is the form in which it naturally occurs in the organism. Sjoqvist^ has
recently estimated the molecular weight of antipeptone by cryoscopic determina-
tion at 250 ; this agrees very closely with the molecular weight similarly
determined by Siegfried for his new acid, and increases the probability that
the two substances are identical.

When a proteid is subjected to tryptic digestion, a portion is decomposed
beyond the stage of albumose or peptone, and there are formed several nitro-
genous bodies of much simpler constitution ; of these, some are amido-acids
and some organic bases. Of these substances, two amido-acids, leucine or amido-
caproic acid, and tyrosine or para-oxyphenylamido-propionic acid, are present in
much larger quantity than the others, which only occur in traces. These others
are aspartic acid or amido-succinic acid, glutamic acid or amido-pyrotartaric
acid, butalanine or amido-valerianic acid ; and of bases, ammonia, lysine, and
lysatinine. Besides these substances of known composition, there is another
substance of unknown composition formed, to which the name of tryptophan
has been given, although it has never been isolated, and is only known through
certain peculiar colour reactions which it gives.

The amido-acids formed in tryptic digestion.-— Leucine. — Leucine
is an amidocaproic acid ((CH3)2CH.CH,.CH(1sTH2).COOH), and is always formed
in any profound decomposition of proteid, such as boiling with dilute acids or
alkalies, fusing with alkalies, in tryptic digestion, or in putrefaction. It has
been found in nearly all the tissues in the body, and there has been much
discussion as to whether it is a normal constituent here, or is formed as a
post-mortem product. Certainly it is rapidly increased in amount, because of
proteid decomposition, after death, but the evidence is strong for its normal
presence in more or less pronounced traces in most of the organs in the fresh
condition. It is, besides, a very common constituent of tissue in many
pathological conditions, and also occurs in the vegetable world.

Yirchow showed that both leucine and tyrosine are found normally in the
pancreas after death, and Kiihne afterwards showed that its amount here was
much increased by auto-digestion of the gland tissue post-mortem.

Leucine was first discovered by Proust in 1818 in putrefying cheese, and
named by him cheese oxide (Kase-oxyd). It was also obtained by Braconnet
by decomposing animal matter with sulphuric acid.^

Leucine may be prepared in many ways : by tryptic digestion of proteids,
by boiling various forms of proteid with dilute acids or alkalies, with stannous
chloride and hydrochloric acid, with bromine water in sealed tubes, or by
fusing with caustic alkalies. A common method is that of boiling horn
shavings with dilute sulphuric acid for many hours ; but any form of proteid
will yield it when so treated, such as meat, cheese, fibrin, wool, feathers,
elastic tissue.

Leucine has been obtained artificially by Limpricht,"^ by acting on isoval-

^ Skandin. Arch. f. Physiol., Leipzig, 1896, Bd. v. S. 277.

2 For a A'ery full accoLUit of these bodies, see Gamgee, " Physiological Chemistry of the
Animal Body," vol. ii. p. 231.

3 Maly, Hermann's "Handbuch," Bd. v. (2), S. 207.
* Ann. de cMvi., Paris, 1854, tome xciv. p. 243.



4 2 2 CHEMISTR Y OF THE DIGEST! VE PR O CESSES.

eraldeliyde with hydrocyanic and hydrochloric acids. Isovaleraldehyde
(C^HgCOH) is prepared, according to the general method, by oxidising amyl
alcohol with potassium bichromate and sulphuric acid ; purified by forming the
sodium bisulphite compound, decomposing this and collecting the distillate;
this is shaken with ammonia, when isovaleraldehyde-ammonia is thrown down
in crystalline form. These crystals are washed Avith water, and then boiled
with a mixture of strong hydrocyanic and dilute hydrochloric acids, when
a reaction takes place yielding a body of the composition C^gHggXg, which
breaks up into leucine and ammonia.

C18H33N5 + 6H,0 = 3(CeHi3NO,)+2 IsTHg.
(leucine)

Leucine has also been obtained artificially by Hiifner,^ by heating mono-
bromocaproic acid with saturated ammonia under pressure to 120°— 130° C
during four or five hours.

C^HioBrCOOH + ^B.^ = G^YL^,{'SB.^)COOB. + HBr.

Constitution of leucine. — That leucine is an amidocaproic acid is shown
both by these methods of artificial preparation and by the following
reactions : —

1. Heated under pressure to 140°-150° C, with strong hydriodic acid, it
yields caproic acid, iodide of ammonium, and iodine.

C,H,o(NH2)COOH + 3 HI = CsHi.COOH + NH^I + \

(leucine) (caproic acid)

2. Heated alone, rapidly over its melting-point (170° C), to 180°-200° C,
it yields amylamine and carbon-dioxide.

C5Hio(ISrH2)COOH = C5Hi,IsTH2 + GO,

3. When acted upon by nitrous acid, it breaks up in the usual manner of
amido-acids, all the nitrogen being evolved as such, and oxycaproic or leucic
acid being simultaneously formed.

C5Hio(N'H2)COOH + HI^O^ = C5H,o(OH)COOH + H^O + N2

(leucine) (leucic or oxj'caproic acid)

These reactions show that leucine is an amidocaproic acid, but there are
several isomeric amidocaproic acids.^ It was thought until quite recently
that leucine was the amido-acid of normal caproic acid, but it has been recently
shown to be amido-isobutylacetic acid.^ The difference in the structure of these
two compounds would be represented according to the usual convention by the
two following graphic formulae : —



IsTormal a-amido-
caproic acid



Pure leucine crystallises in the form of thin white transparent plates,
forming in mass a snow-white powder, which feel greasy and are not wetted

^ Chem. Genlr.-Bl., Leipzig, 1869, S. 159; Journ. f. 'praM. Ghem., Leipzig, 1870,
Bd. i. S. 6.

^ According to R. Colin, not one but several leucines are formed in pancreatic digestion ;
these are probably the isomeric amidocaproic-acids, Ztschr. f. pluysiol. Chem., Strassburg,
1894, Bd. XX. S. 203.

^ Schulze and Likiernik, Bcr. d. dentsch. chcm. Gesellsch., Berlin, 1891, Bd. xxiv. S. 6G9 ;
B. Gmelin, Inaug. Diss., Tubingen, 1892.



CH3




CH3 CH3


CH2




\/


CH2


Isobutyl-(a) amidoacetic


CH


CH2


acid, or leucine


CH.,


CH— NH2




CHNH


COOH




COOH



AMID 0-A cms FO RMED IN TR YPTIC DIGESTION. 4 2 3

by water, so that they float on its surface, although their specific gravity is
about 1 "3 ; but usually leucine is found to separate from solutions containing
it in characteristic globules of microscopic size, often exhibiting a radial
striation, or a marking off into concentric alternately dark and light bands.
In this latter impure form it is easily soluble in water, and fairly so in
alcohol ; the pure product is less soluble, its solubility is variously stated from
1 in 29 to 1 in 47 parts of water at room temperature. This difference is
usually ascribed to the presence of different isomeric modifications in varying
proportions.

Heated slowly to 170° C, leucine melts and commences to sublime in loose
woolly flocks, resembling those formed when zinc is burnt to zinc oxide ; these
present the appearance microscopically of thin plates grouped into rosettes.
Leucine is very feebly soluble in strong alcohol (about 1 in 1000 in 98 per cent,
alcohol), and is insoluble in ether.

The artificial leucine obtained as described above is inactive ; so is that
obtained by the action of barium hydrate on proteids at high temperatures
(150°-160° C). Leucine from the tissues is dextrorotatory, but also becomes
inactive when heated to 150° C. with baryta water. When Penicillium
glaucum is sown in inactive leucine, the organism lives on the dextrorotatory
variety, and laevorotatory leucine is left behind. These two are physical isomers
of each other; their specific rotatory powers are (a)-D= + 17"3 for the right-
handed, and (a)-D= - 17"5 for the left-handed.^

Tests for leucine. — Leucine may be recognised —

1. By its crystalline form in the above-described spherules, forming from
solution, and yielding a woolly sublimate which shows rosettes of platelets
under the microscope. If it be heated rapidly so as to raise the temperature
much above 170°, in subliming it the odour of amylamine is obtained.

2. By dissolving in boiling water and adding boiling solution of cupric
acetate, when a deep blue coloured crystalline compound apjDears.

3. By Scherer's test, which consists in adding a drop of nitric acid and
slowly evaporating on platinum foil, when a nearly colourless residue is left.
If this be wetted with sodium hydrate and gently heated, it forms into an oily
globule which rolls about on the foil.

Tyrosine. — Tyrosine, or para-oxyphenyl-a-amidopropionic acid (CqH4(0II)
CH2CH(NH2)COOII), is the almost constant companion of leucine in the
decomposition of proteids. Unlike leucine, tyrosine is never found as a con-
stituent of fresli tissues ; its supposed presence in fresh pancreas has been
shown to be due to self-digestion of the gland,^ and it is not found in other
fresh tissues, but is a constant constituent of those in which proteid decom-
position has set in. It occurs very plentifully in old cheese, from which it
was first obtained by Liebig by fusing with caustic potash.

Tyrosine may be obtained in general by the same methods as leucine, but
it is not formed in the decomposition of gelatin nor of antipeptone.

Gonstitution of tyrosine. — The constitution of tyrosine has been established
mainly by the work of von Bartli,^ who first showed that tyrosine yielded on
fusing with caustic potash ^a7'a-oxybenzoic acid, an isomer of salicylic acid.
Previously to this, tyrosine had been looked upon as a derivative of salicylic
acid, but from this von Barth concluded it must be ethylamido-^rwa-oxybenzoic
acid (CgHj.ISrH.CgHg.OH.COOII). If this formula were correct, on treating
with hydriodic acid, ethylamine (C2HJJ.NH2) ought to be obtained, but Htlfner
showed that ammonia instead was split off. Yon Barth next found that the

^ Schulze and Boshard, Ztschr. f. physiol. Chem., Strassburg, 1885, Bd. ix. S. 63 ; 1886,
Bd. X. S. 134.

^ Radziejewski, Virchoio's Arclnv, 1866, Bd. xxxvi. S. 1; Kilhne, Untersuch. a. d.
physiol. Inst. d. tfniv. Heidelberg, Bd. i. S. 317.

'Ann. d. Chem., Leipzig,, Bd. cli. S. 100. See also Erlenmeyer u. Lipp, Ber. d. deutsch,
chem. Gesellsch., Berlin, 1882, Bd. xv. S. 1544.



42 4 CHEMISTR Y OF THE DIGESTIVE PROCESSES.

NHg group in his reaction was not replaced by hydrogen but by hydroxyl,
and so finally arrived at the formula CqH^.OH.CoH3(NH2)COOH, which is
in agreement with all the experimental facts, and is now universally accepted.

When fairly pure, tyrosine crystallises in long slender needles, which occur
both singly and in double sheaves or in rosettes. If impure, however, it very
often separates in balls or nodules closely resembling those of leucine,
recrystallising from Avarm water in the crystalline form above described ; if
the solution containing the crystals be filtered, these felt themselves together
on the surface of the paper to a thin, snow-white, paper-like mass. Tyrosine
is much more insoluble in water than leucine (1 in 1900 of cold water), more
so (1 in 150) in boiling water and in dilute and concentrated mineral acids,
and also in alkaline solutions (ammonia, alkalies and their carbonates, and the
alkaline earths). Tyrosine exhibits the usual facility of amido-acids for
forming compoimds, both Avith bases and acids ; the copper compound is
sparingly soluble in water, and is formed in dark blue needles on the addition
of freshly precipitated cupric hydrate to a boiling solution of tyrosine, and
allowing to cool.

Tyrosine, unlike leucine, cannot be sublimed without decomposition, and on
dry distillation yields carbon-dioxide and a base of the composition CgH-^jNO.

Tests for tyrosine. — Tyrosine may be identified by the following tests : —

1. Its crystalline form.

2. Scherer's test, which consists in evaporating a portion with strong nitric
acid in a platinum dish, leaving a transparent deejD yellow residue, which
turns red on moistening with caustic soda solution, and then a blackish brown
on again evaporating.

3. Piria's test. — A drop or two of strong sulphuric acid is added to the
tyrosine in a watch-glass ; after half-an-hour, during which tyrosine sulphuric
acid forms, the acid is diluted with water, and neutralised by the addition of
calcium carbouate. The solution is filtered from the calcium sulphate so
formed, and a drop of neutral ferric chloride solution added, when a deep
violet colour appears, similar to that given by salicylic acid.

4. R. Hoffmann's test. — This is really identical with the Millon test for
proteids, and in cases where there is no group present in the proteid molecule
capable of yielding tyrosine, the test with Millon's reagent does not succeed,
e.g., in the case of gelatin and of antipeptone. The test may be carried out
directly in the case of tyrosine itself, by boiling a solution containing this Avith
Millon's reagent, when the solution passes through pink into deep crimson.

Separation of leucine and tyrosine. — Leucine and tyrosine may very easily
be separated Avhen in solution together by means of their very different
solubilities. To separate them after pancreatic digestion, it is best to allow
digestion to j^roceed for several days ; at the end of this time there is no
coagulable proteid, or albumose, except in traces, left in the solution. This is
neutralised and evaporated down, when the tyrosine, on account of its sparing
solubility in Avater, is throAvn out in crystalline masses, Avhile the more soluble
leucine nearly all remains in solution ; on cooling, more of the tyrosine separates
out, and when the solution is cold it is filtered otF, extracted Avith hot alcohol
to remove traces of leucine, and purified by recrystallisation from hot Avater,
or by dissolving in weak ammonia and precipitating by neutralisation.

The filtrate containing the leucine and peptone is still further evaporated
until it becomes syrupy ; it is then extracted with boiling alcohol, which takes
up only the water and leucine. On evaporating off the alcohol, leucine is thrown
out of the concentrated solution, and may be purified by sublimation or by
repeated recrystallisation from alcohol. More tyrosine may be obtained from
the residue left by the boiling alcohol.

Or the solution, after completion of digestion and careful neutralisation,
may at once be evaporated to a thin syrup and set aside for tAventy-four hours.



AMIDO-A cms FORMED IN TR YPTIC DIGESTION. 425



during which time most of both the leucine and tyrosine crystalHses out. After
separation of the crystals, the filtrate may be once more reduced in bulk by
evaporation and a second crop of crystals obtained as before.

To the syrupy mother-liquor now remaining absolute alcohol is added, until
precipitation of the peptone commences, when the addition of alcohol is
stopped and the precipitate of peptone redissolved by gently Avarniing. The
solution is now set aside to cool and crystallise as before. The united crops of
crystals of mixed leucine and tyrosine are boiled with alcohol, which dissolves
the leucine and but little of the tyrosine. On concentrating this alcoholic extract,
leucine crystallises out and may be purified by recrystallisation from alcohol.
From the residue insoluble in alcohol the tyrosine is obtained by dissolving in
weak ammonia water and neutralising.

The yield, both of leucine and tyrosine, obtained from different materials
varies greatly, but in all cases the former is always formed in much larger
quantity. The following table ^ gives the percentage yield of the substances
obtained in some cases ; the figures indicate parts per 100 : —



Source.


Leucine.


Tyrosine.


Observers and Method.


Gelatine .


1-5-2 («)


None (a)


(a) NencJci, boiling with dilute
sulphuric acid.


Ligamentum nucliae


36-45 (&)


0-25 (&)


(&) HrlenmeyersindSchdffer, boil-
ing for some hours 1 pint of
material, 2 pints sulphuric
acid, 3 pints water.


Fibrin


14-0 (6)


2-0 (&)-3-3 (c)


(c) Hlasiwetz and Rabermann,
heating with bromine un-
der pressure.


Muscle .


18-0 {h)


1 -0 (/.)


(d) StUdeler, heating with sul-
phuric acid.


Horn


10-0 (&)


3-6 (&)-4-0((^)


(e) ScJmtzenberger, heating with
baiyta water for four to six
days, at 160°-200° C.


Egg albnniin .


22-6 (c)


1-0 (&)-2-0 (c)




Plant albumin


] 7 -3 (c)


2-0 (e)




Casein


19-1 (c)


4-1 (c)




Fibrin .


7-9 (/)


3-3 (/)


(/) Kuhne, digestion of boiled
fibrin.



Aspartic acid, or amido-succinic acid [C._,H3.(NHo).(COOH)2], does not
occur in any of the animal tissues or secretions, but is formed in small
quantity in all those decompositions of proteids and their allies already described
as furnishing leucine and tyrosine.^ It was first identified among the products
of pancreatic digestion of fibrin by Radziejewski and Salkowski,^ and von
Knieriem afterwards showed that it is also formed in the pancreatic digestion
of plant glutin.

It may also be obtained by decomposing asparagin (amido-succinamic acid)
by an alkali or acid, thus : —



CH2— COOH



CH(NH2)— C0(NH2) + HCl + H,0

(asparagin or amido-succinamic acid).



CH.— COOH

I ^ -flsTH^CL

CH(NH2)— COOH

(aspartic acid or amido-succinic acid).



1 Compiled from Maly, Hermann's "Handbuch," Bd. v. (2), S. 209 ct seq.

^ Ritthausen and Kreuster, Joiirn. f. prakt. Chcm., Leipzig, 1871, Bd. iii. S. 314;
Hlasiwetz and Habermann, Ann. d. Chem., Leix)zig, 1871, Bd. clix. S. 304.

2 Radziejewski and E. Salkowski, Ber. d. deutsch. chem. Gesellsch., Berlin, 1874, Bd. vii.
S. 1050 ; Ann. d. Chem., Leipzig, 1873, Bd. clxix. S. 150 ; W. v. l^nieriem, Ztschr. f. Biol.,
Munchen, 1875, Bd. xi. S. 198. From 100 pts. of dry egg albumin Hlasiwetz and
Habermann obtained 23-8 pts. of aspartic acid by the action of bromine in sealed tubes.



426 CHEMISTR V OF THE DIGESTIVE PROCESSES.

Aspartic acid is soluble with difficulty in cold water, easily soluble in boiling
water, and insoluble in alcohol. It crystallises in rhombic prisms ; its solutions
are optically active, and curiously Avhen in acid solution it is dextrorotatory,
but laevorotatory when in alkaline solution. It forms a crystalline compound
with copper, which may be used for purifying it. After leucine and tyrosine
have crystallised out from the products of a proteid decomposition, they
are separated from the mother-liquors, and these are further concentrated
and treated with a small quantity of alcohol, when after a time a neAv
crust of crystals forms. These are dissolved in water, the solution is
boiled with freshly precipitated cupric hydrate and filtered ; in the filtrate,
on cooling, crystals are deposited of the copper salt of aspartic acid just
mentioned. These crystals are dissolved in hydrochloric acid, the copper
is thrown oiit by a stream of sulphuretted hydrogen, and the copper
sulphide filtered off ; in the filtrate, crystals of aspartic acid separate



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