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Ztschr. f.physiol. Chem., Strassburg, 1883, Bd. viii. S. 129; Hasebroek, ihid., 1887, Bd.
xi. S. 348 ; A. Herrmann, ibid., 1887, Bd. xi. S. 508 ; Neumeister, Ztschr. f. Biol.,
Mlinchen, 1890, Bd. xxvii. S. 310.

^ Arch, de physiol. norm, etpaih., Paris, 1893, tome xxv. p. 447.

^ Verhandl. d. naturh.-med. Ver. zu Heidelberg, 1877, JST. F., Bd. i. S. 236.

^ Vide infra. s Loc. cit.



4o6 CHEMISTR Y OF THE DIGESTIVE FEO CESSES.

closely resembling Meissner's parapeptone antialhumate ; considering
them anti bodies, from the fact that they do not yield leucine and
tyrosine on tryptic digestion, but are, though with difficulty (especially
in the case of antialbumid), converted into antipeptone.

Ktihne gave the following graphic representations of the cleavage of
proteids by acids and by digestion : — •

Scheme of Proteid Cleavage hy Acids.

ALBUMIN.



Autigroup. Hemigronp.

Antialbumid.

Antialbumate.

Antialbumose. Hemialbumose.

Antipeptoue. Hemipeptone.



Scheme of Digestive Cleavage of Proteids.

ALBUMIN

(Albuminate).



Autialbumose. Hemialbumose.



Peptic
Digestion, j



II I I ^ j.rypuG

^ Antipeptoue. Antipeptone. Hemipeptone. Hemipeptone. Digestion.



Tryptic



Leucine. TjTOsine. Leucine. Tyrosine,



;



Klihne, in conjunction with Chittenden,^ subsequently investigated
more minutely the intermediate products in peptic digestion, and those
formed by the action of dilute acids. The following is an account of
the substances obtained and their mode of preparation : —

Antialbumid. — This substance was prepared as follows : — The white of fifty
eggs, freed from membrane and much diluted, Avas made feebly acid with,
sulphuric acid and coagulated by boiling. The coagulum was suspended in
1300 c.c. of Avater containing 7 c.c. of sul2:)huric acid and heated to 100° C. ; after
ten hours it appeared little altered and was filtered off. The filtrate gave on
neutralisation a precipitate principally composed of acid albumin. After
removal of the first acid, the albumin which had remained undissolved was
heated Avith 3 litres of ^ per cent, sulphuric acid to 100° C. for nineteen hours,
then collected on a filter and completely Avashed.

The albumid thus obtained AA^as insoluble both in dilute and concentrated
acetic acid, and in hydrochloric acid of 1 "4 per mille and stronger, but easily
soluble in dilute caustic soda solution and in dilute alkaline carbonates, from
Avhich it AA'as precipitated by concentrated sodium chloride. Purified by diges-
tion Avith gastric extract and 4 per mille hydrochloric acid for six hours at
40° C, it remained undissolved, but changed in appearance, becoming clotlike.
The clot Avas washed Avith Avater, dissolved in 1 per cent, solution of sodium
carbonate, filtered, reprecipitated Avith sulphuric acid, and Avashed again. It
noAV dissolved in 2 per mille hydrochloric acid, and in this solution Avas digested
with good peptic extract for eighteen hours. It Avas unchanged, and reappeared,
in equal amount to the eye, on neutralisation of the solution.
1 Ztschr.f. Biol., Muncheii, 1883, Bd. xix, S. 159.



CLE A VA GE THE OR Y OF PRO TEW DIGESTION. 40 7

Now washed with water until no reaction for chlorides was obtained, and
afterwards treated with alcohol and ether, it formed a powder of slightly
yellowish colour. A part of this purified antialbuniid was dissolved in sodium
carbonate solution of "5 per cent., and treated with a dialysed and very active
trypsin solution at 37°-38° C. After thirty minutes the mixture began to be
turbid, and in two hours solidified to a clot. By breaking up the clot and
filtering, the fluid part was separated from the clot; it was made scarcely
turbid by neutralisation, and yielded by further digestion no new precipitate, but
contained a fair amount of peptone.

The separated clot was soluble in hydrochloric acid of 2 per mille, but as
insoluble in sulphuric acid of 4 per mille as the original precipitate. It was
completely precipitated from solution in 1 per cent, sodium carbonate solution
by concentrated sodium chloride solution. From this it seems that in the
coagulation of albumid in trypsin no change takes place other that its becom-
ing more insoluble in sodium carbonate solution. The action of trypsin in
more alkaline solution was next tried; after the first precipitation of the
albumid it was dissolved in -75 per cent, sodium carbonate solution, digested
with dialysed tryptic fluid, neutralised and filtered. The clotlike albumid was
dissolved in 5 j)er cent, sodium carbonate solution, and by repeated digestion
with dialysed tryptic fluid, the greater portion was converted into antipeptone.
The final residue from this much accentuated tryptic digestion was completely
insoluble even in 5 per cent, sodium carbonate solution, but dissolved in 1 per
cent, caustic soda. Precipitated by neutralising with hydrochloric acid,
washed, redissolved in sodium carbonate, and treated with trypsin anew, it was
again completely thrown out as a clotlike coagulation. ISTo leucine or tyrosine
was j)resent in the tryptic filtrates.

How widely this account differs from the statement which occurs in
most text-books, that antialbuniid is not attacked by pepsin, but is
converted into antipeptone by trypsin, may easily be seen. In a fluid of
equal alkalinity to that found in the body, antialbuniid is no more
digested by trypsin than it is by pepsin and hydrochloric acid. Now
it has been shown that trypsin is most active in a sodium carbonate
solution of about 1 per ceiit.,^ and considerably less active in one of 5
per cent. ; why then does trypsin in 5 per cent, solution do that wdiich it
is unable to do in 1 per cent, solution ? Obviously because the 5 per
cent, solution dissolves the clot of antialbuniid, while the 1 per cent,
solution does not. In the former case a weaker trypsin acts on anti-
albumid in solution ; in the latter, a stronger trypsin on antialbuniid as
an insoluble precipitate.

Be this as it may, antialbumid is only with great difficulty and
incompletely peptonised by trypsin. In all its properties, from its mode
of formation onward, the substance appears to be merely a very insoluble
form of acid albumin.

Antialb'umose. — By a fractionated peptic digestion, Kiiline and
Chittenden 2 obtained a substance which they termed antialbumose.
The preparation of this substance from white of egg is as follows : —

The white of fifty eggs was freed from membrane, diluted and coagulated
by boiling after acidifying with acetic acid. The coagulated proteid was
digested in two litres of 4 per mille hydrochloric acid and one litre of dialysed
gastric extract for one and a half hours at 40" C, it was then allowed to cool
to the temperature of the room and filtered from the undissolved part, the
process of filtration occupying two days. The undissolved residue was again
treated Avith fresh gastric extract until it was all dissolved, which occupied
1 See p. 338. " Loc. cit., p. 171.



4o8 CHEMISTR Y OF THE DIGESTIVE PROCESSES.

fifteen hours. After filtration the fluid was neutralised, and the neutralisation
precipitate separated. This precipitate was digested anew for forty-eight
hours with 150 c.c. of strong gastric extract, after which it was re-obtained on
neutralisation not sensibly diminished in amount. Dissolved in '75 per cent,
sodium carbonate, and mixed with powerful dialysed pancreatic extract, it gave
no clot (see " Antialbumid ") when kept for forty-eight hours at 40° C, and
neutralisation precipitated only a part, the rest being converted into peptone.
This last neutralisation precipitate showed the properties of antialbumid ;
dissolved in sodium carbonate solution of 1'8 per cent., it was clear at first, but
began to cloud in one to two hours, and in twenty-four hours about half had
set into a thick clot which could not be peptonised completely by either peptic
or tryptic digestion. The various pancreatic solutions separated from the
neutralisation precipitates contained only peptone, and were free from leucine
and tyrosine. The antipeptone here obtained contained 30 per cent, of ash.

Of these three auti-compounds it is only claimed that one, anti-
albumose, is a product of natural digestion ; the other two, antialbumid
and antialbumate, are admittedly products of acid action or of acid and
vei'y weak peptic solution, which amounts to the same thing, — and the
fact that they cannot be converted into peptones by the prolonged and
repeated action of pepsin and hydrochloric acid proves that they are not
natural products of strong peptic digestion in which no such inconvertible
residue is formed. Antialbumose is commonly stated to be convertible
by prolonged peptic digestion into peptone ; but, as may be seen from
the above description, it is not materially altered by forty-eight hours'
digestion with a strong extract of gastric mucous membrane, and even
with trypsin a considerable portion is left unaltered, betrapng all the
properties of antialbumid. Antialbumose possesses all the chemical
properties of an acid albumin and none of those of the albumose class, so
that its name is a misnomer ; no such substance as an antialbumose has
actually been isolated. Antialbumid, antialbumate, and antialbumose,
to place them in the order of their solubility and facility for under-
going decomposition, are three substances all of which are remarkably
resistant to both peptic and tryptic digestion, and belong more to the class
of acid albumins than to any other. It is now generally recognised that
acid albumin is a generic and not a specific term, and it is to be hoped
that room will soon be found for these three bodies in this class, and the
terminology of digestion left a httle less complicated than it is at present.
It may he asked, AVhy was antialbumose, if it is not a natural product
of peptic digestion, obtained in the above experiment ? The authors
themselves remark on the close resemblance between their product
and Meissner's parapeptone. The latter is produced either by the action
of dilute acid or of a very weak pepsin solution in the presence of acid.
Now for two days, while filtering at atmospheric temperature, after the
first hour and a half of digestion, the substance was under exactly the
proper conditions for the production of parapeptone. Finally, no product
so resistant to both pepsin and trypsin, as this substance is shown to be
by the above description, is formed during uninterrupted digestion.

Another method for preparing ^'antialbumose." — Kfihne and Chittenden^
also prepared antialbumose from fibrin by a somewhat similar course of
procedure, except that there was here no tAvo days' delay in filtering, since the
fibrin was more quickly dissolved. There is, however, an objection no less
fatal, as will be pointed out after a description of the process.

■* Loc. cii.



CLE A VA GE THEOR Y OF PROTEID DIGESTION. 409

Five hundred grms. of unboiled fibrin, squeezed as dry as possible with the
hand, were placed at room temperature for twenty-four hours in 5 litres of 0*2
per cent, hydrochloric acid ; the mixture was then heated to 37° C, and
100 c.c. of gastric extract added. Solution took place inside an hour, after
which the fluid was filtered through a hair sieve, digestion stopped by neutral-
isation, and the neutralisation precipitate filtered off'. This precipitate is stated
to be essentially antialbumose. It was long washed with water, and did not
then dissolve easily in 0*2 per cent, hydrocldoric acid, so was heated for some
hours at 40° C. This acid solution was treated with an equal volume of strong
gastric extract in 0-2 per cent, hydrochloric acid for forty-eight hours, again a
heavy neutralisation precipitate was obtained. This precipitate, after washing
with water thoroughly till no biuret peptone reaction was given, was treated
with sodium carbonate solution of 2*5 p)er cent., in luhich it teas not easily soluble,
and the solution was not clear until it had been digested for forty-eight hours at
48° C. with trypsin. Even then, on neutralising, a precipitate behaving like
antialbumid Avas obtained. Eedissolved in 2-5 per cent, sodium carbonate solu-
tion, and redigested with trypsin, it was again precipitated in clotlike flakes,
and was only very slowly and partially converted by repeated tryptic digestion.

Here, again, there is no guarantee, after heating the first neutrahsation
precipitate for some hours with 0-2 per cent, hydrochloric acid in order
to dissolve it, that a natural digestion product remains to be dealt with
in the subsequent processes. In addition, the obstinate resistance of the
substance to both peptic and tryptic digestion proclaims it a product of
experimental procedure, and not a true stage in natural or uninterrupted
digestion.

Hemialbumose. — Iviihne and Chittenden ^ also obtained a precipitate, to
which they gave the name of hemialbumose ; this was obtained from the pro-
ducts of fractional peptic digestion in the filtrate after the removal of the
so-called antialbumose by neutralisation. This filtrate was concentrated to one-
fourth of its volume, acidified with acetic acid, boiled and filtered from a scanty
coagulum, again concentrated and precipitated by the addition of excess of
alcohol. In this precipitate by alcohol, the authors recognised, besides peptones,
two forms of albumose, soluble and insoluble hemialbumose. The precipitate
was rubbed up with cold water, until the wash water no longer gave the biuret
reaction. A part of the albimiose (soluble hemialbumose) went into solution,
accompanied by all the peptone, a part remained insoluble (insoluble hemi-
albumose). The latter substance was not pure, but contained a proteid substance
insoluble in 2 per cent, acetic acid and in sulphuric acid of 0*4 per cent., and
with difficulty soluble in dilute caustic soda solution. The " insoluble hemialbu-
mose " was separated from this by treating with boiling water. From solution
in boiling water a part of the "insoluble hemialbumose" was precipitated as
the solution cooled. This was separated ; the remainder Avas precipitated from
the cold solution and added to it. The "soluble hemialbumose " was obtained,
free from its admixture with peptone in the cold water extract, by Salkow^ski's
method of boiling with excess of sodium chloride and dilute acetic acid so as
to form a saturated solution, washing the precipitate with saturated sodium
chloride solution, dissolving in water and dialysing until the dialysate gave
no reaction for chlorides with silver nitrate.

These hemialbumoses on tryptic digestion yielded leucine and tyrosine
abundantly, but could not be completely broken up by such digestion,
a variable amount of peptone being always left, no matter how prolonged
the digestion, which could only (on the cleavage theory) be antipeptone,
and so pointed to impurities in the form of anti-compounds in these

^ Loc. cit.



4IO CHEMISTR Y OF THE DIGESTIVE PROCESSES.

hemialbiimoses (or otherwise to the non-existence of cleavage at the
albumose stage into hemi and anti groups). ISTor, when these hemi-
albumoses were subjected to more prolonged digestion yielding hemi-
peptone (?), could this substance be completely broken up by prolonged
tryptic digestion.

Kiihne ^ also described as hemialbumose a substaBce occasionally found in
the urine of patients suffering from osteomalacia, and first discovered by
Bence Jones. Much has been made of the importance of this albnmose by
the supporters of the cleavage theory, but there is no more evidence that it is
a pure hemialbumose than there is in the case of the substances described
above ; that is to say, it has not been shown to be completely broken up by
tryptic digestion, and this is the crux of the whole question. The fact that it
yields leucine and tyrosine proves nothing. It has not been experimentally
shown that no peptone is left after the prolonged action of trypsin upon it.

Separation of the various alhumoses from the " hemialbumose " ^jre-
ciijitate. — Stimulated by a desire to obtain a pure hemialbumose which
should be capable of complete decomposition past the peptone stage by
trypsin, and encouraged in the belief that hemialbumose was a mixture,
as well by the known existence of two physically different forms (the
soluble and insoluble described above) as by certain inconstancies in its
behaviour tow^ards sodium chloride, Kiihne and Chittenden ^ set to work
again upon the subject, and although they did not quite achieve their
object, produced a research which, whether the cleavage theory stands
or falls, must, from the experimental point of view, always remain of the
highest A^alue, containing as it does the first basis for a classification of
the albumoses, the first light cast upon the relationship of this class
of proteids.

From the hemialbumose described in their previous paper, they were
able to separate, by the action of sodium chloride under various
conditions, four substances with the following properties : —

1. Protoalbumose. — Precipitated by saturation with sodium chloride,
soluble in cold and hot water.

2. Heteroalbumose. — Also precipitated by saturation with sodium
chloride, but insoluble in cold and in boiling w^ater ; soluble in dilute and
in moderately concentrated saline solution.

3. Dysalbumose. — The same as heteroalbumose, but insoluble in
saline solution. This solution was recognised to be merely a more
insoluble modification of heteroalbumose ; each of the two substances is
easily convertible into the other. Dysalbumose corresponds to the
" insoluble albumose " of the earlier paper.

4. Deuteroalhumose is not precipitated by saturation with sodium
chloride alone, but is precipitated by saturation with sodium chloride in
the presence of acetic acid, and is soluble in water.

These various albumoses were subjected to tryptic digestion, and it
was found that none was a pure hemialbumose, — all yielded more or less
unconvertible peptone accompanied by leucine and tyrosine. A bigger
yield of amido-acids was obtained from protoalbumose and deutero-
alhumose than from heteroalbumose ; indeed, the latter showed itself to
be more an anti- than a hemi- body, while protoalbumose yielded very
little peptone and an abundance of amido-acids. After this evidence

1 Ztsehr. f. Biol, Mtinclicii, 1883, Bd. xix. S. 209.
' Ihicl, 1884, Bd. xx. S. 11.



CLE A VA GE THEOR Y OF PROTEID DIGESTION. 4 1 1

the term hemialbumose, as applied to the substance, or rather mixture
of substances, described above, ought to have speedily disappeared;
unfortunately it has not yet done so.

Soon after this a valuable aid to the study of the albumoses was
found in the discovery of Wenz,^ that saturation with ammonium
sulphate precipitated all albumoses from solution, while the peptones
remained dissolved. Heynsius^ first noticed the powerful action of
ammonium sulphate as a proteid precipitant, but fell into error in
thinking that it precipitated peptones as well. More careful experiments
by Wenz, in Klihne's laboratory, showed that it did not precipitate
peptones, and so it was instituted as a means of separating albumoses
and peptones. The statement, however, that saturation with ammonium
sulphate totally precipitates albumoses and leaves peptones dissolved,
can only be macle with a certain reservation. Certain proteid sub-
stances remain unprecipitated by saturation with ammonium sulphate,
and these may conventionally be labelled peptones; but it has been
shown ^ that, in order to precipitate completely bodies which had
been known as albumoses before the introduction of ammonium sul-
phate, it is necessary to help the ammonium sulphate by saturating in
dilute solution and with varying reaction. If these bodies had not
been classed with the albumoses before Wenz's discovery, they would
probably now be peptones ; so conventional and artificial as this is the
proteid classification with which at present we are forced to be content.
In little or nothing except unimportant physical differences are the
albumoses and peptones distmct. If ammonium sulphate did not exist, it
would be difficult to say how to draw a sharp line between them ; * both
classes of bodies give the same reaction to the biuret test, and both are
diffusible, though the albumoses more slowly so than the peptones.^

Se'paration of alhumoses and pe2Jtooies. — The following is the method
recommended by Kuhne ^ for separating albumoses from peptones : —

The fluid containing the products of digestion is freed from albuminates
and coagulable proteids in the usual manner, and then, when sufficiently diluted
and of nearly neutral reaction, is saturated while boiling with ammonium
sulphate, and separated on cooling from the excess of salt and precipitated
albumose. The solution is again heated, and after it commences to boil it is
macle strongly alkaline by the addition of ammonia and ainmonium carbonate,
then again saturated with ammonium sulphate, and once more allowed to cool,
when a second precipitation of albumose and excess of salt takes place. A
third time heated, until the smell of ammonia disappears, it is once more
saturated while warm and made decidedly acid in reaction by the addition of
acetic acid, Avhen, on cooling, a third and last precipitation of albumose takes
place, and the filtered fluid is supposed to contain nothing proteid except
peptone ; amphopeptone if the original fluid was the result of gastric digestion.
The albumoses can be obtained by dialysis and concentration from the united
precipitates.

'^Ztschr.f. Biol, Miinchen, 1886, Bd. xxii. S. 1.

2 Arch./, d. ges. Physiol., Bonn, 1884, Bd. xxxiv. S. 330.

SKlihne, Ztschr. f. Biol., Miinchen, 1893, Bd. xxix.

^For a discussion of this point see Pekelharing, Arcli. f. d. gcs. Physiol., Bonn, 1880,
Bd. xxii. S. 185 ; 1881, Bd. xxvi. S. 515 ; Internat. Beitr. z. wissensch. Med. Festschr. R.
Virchow . . ., Berlin; Ztschr. f. Biol., Miinchen, 1891, Bd. xxviii. S. 567; JSTeumeister,
ibid., S. 361 ; Kiihne, iUd., S. 571.

5 See Kiihne, Ztschr. f. Biol., Miinchen, 1892, Bd. xxix. S. 20; Chittenden and
Amerman, Journ. Physiol., Cambridge and London, 1893, vol. xiv. p. 483.

^ Loc. cit.



4 1 2 CHEMISTR Y OF THE DIGESTIVE PR O CESSES.

The amphopeptone is obtained from the filtrate after removal of the
ammonium sulphate by complicated methods, consisting essentially in the
removal of ammonium sulphate as far as possible by concentration ; solution
of the amphopeptone in weak alcohol; removal of as much ammonium
sulphate from the weak alcohol as possible by a freezing mixture ; removal of
the alcohol by distillation ; removal of the last portions of ammonium sulphate
by boiling with barium carbonate ; removal of the last traces of barium salts by
cautious addition of dilute sulphuric acid ; and finally, precipitation of the
amphopejDtone by excess of absolute alcohol.

Neumeisters method for sejjarating the alhumoses of ^jeptic digestion.
— The method of Kiihne and Chittenden for the separation of the
various albumoses has been perfected by Neumeister,^ who has in
addition proved that these bodies are not formed synchronously in the
process of digestion, or other form of hydrolysis, but that there are two
stages in the process. In the first stage p7vto- and /le^^e^^oalbumoses are
formed, which are called for this Yeason po-imary albumoses ; in the second
stage each of these primary albumoses gives rise to a deuteroalbnmose,
and these deuteroalbumoses are hence called secondary albumoses.

Since heteroalbnmose is completely and protoalbumose only partially
precipitated by saturation with sodium chloride in neutral solution,
while deuteroalbnmose is not precipitated at all, it is easy, from a
mixture of all three albumoses, to obtain a solution containing only
heteroalbumose and protoalbumose; and on dialysis of this solution
heteroalbumose, being insoluble in water, is precipitated alone, leaving
in solution only pure protoalbumose. In this way pure proto- and
heteroalbumoses can be obtained, but the preparation of pure deutero-
albumose is not quite so easy. In the filtrate from saturation with
sodium chloride there is not only deuteroalbumose but the unprecipitated
residue of the protoalbumose, and on adding acetic acid this is thrown
out along with the deuteroalbumose. However, a loophole is left in the
fact that just as saturation in neutral solution does not precipitate all
the protoalbumose, so saturation in acid solution does not precipitate all
the deuteroalbumose. Neumeister took advantage of this, sacrificed
the first portion of deuteroalbumose thrown out by the acetic acid,



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 58 of 147)