is a large excess of proteid matter present,[169] which obviously
means that the acid in such case exists wholly as combined acid.
Indeed, experiments made in my laboratory have shown that as soon as
free acid, especially hydrochloric acid, is present in a solution
containing trypsin, then proteolytic action is at once stopped. When,
however, acids, especially organic acids, are present in a digestive
mixture containing an excess of proteid matter, so that the solution
contains no free acid (or better, with the proteid matter only
partially saturated with acid) then trypsin will continue to manifest
its peculiar proteolytic power, although to a considerably lessened
extent. Hence, it is evident that the ferment may exert its digestive
power under the three possible sets of conditions which, under varying
circumstances, frequently prevail in the small intestine.
[169] Mays: Untersuchungen aus d. physiol. Institute d. Universität
Heidelberg, Band iii., p. 378; also Langley: On the Destruction of
Ferments in the Alimentary Canal, Journal of Physiology, vol. iii.,
p. 263.
In considering the general phenomena of proteolysis by trypsin, one
is especially impressed by the large and rapid formation of peptone
which almost invariably results from the action of a moderately strong
solution of the ferment, on nearly every form of proteid matter. To
be sure, primary products are first formed, but these are quickly
converted into peptone, and a little experience in studying the
action of pepsin and trypsin soon reveals the fact that the latter is
especially a peptone-forming ferment. In other words, it is peculiarly
adapted to take up the work where it has been left by pepsin and, if
necessary, carry forward the hydrolytic change even to the extent of a
conversion of the entire hemi-moiety into crystalline products.
The primary products of trypsin-proteolysis, however, are not exactly
identical with those formed by pepsin. Thus, protoproteoses and
heteroproteoses seldom appear in an alkaline trypsin digestion; the
proteid matter being in most cases, at least, directly converted into
soluble deuteroproteoses,[170] which are then transformed by the
further action of the ferment into peptones and other products. Hence,
we may express the order of events in the trypsin digestion of a native
proteid as follows:
Native proteid.
|
Amphodeuteroproteoses.
|
Amphopeptones.
╱ ╲
╱ ╲
Antipeptone. Hemipeptone.
╱ | ╲
╱ | ╲
Leucin. Tyrosin. Aspartic acid, etc.
[170] R. Neumeister: zur Kenntniss der Albumosen. Zeitschr. f. Biol.
Band 23, p. 378.
In the digestion of fresh blood-fibrin with trypsin, there is
plainly a preliminary solution of the proteid without any marked
transformation or cleavage occurring, the soluble product being
apparently a globulin, coagulating at about 75° C.,[171] viz., at
approximately the same temperature as serum-globulin. This body,
however, quickly disappears, giving place to true deuteroproteoses as
the ferment-action commences; for it is not probable that this globulin
is a product of enzyme-action, but rather represents a simple solution
of the fibrin by the alkaline fluid and salts. In any event, this
globulin-like substance is not formed in the pancreatic digestion of
coagulated-albumin, serum-albumin, or vitellin, and hence cannot be
considered as a true product of trypsin-proteolysis.
[171] Jac. G. Otto: Beiträge zur Kenntniss der Umwandlung von
Eiweissstoffen durch Pancreas-ferment. Zeitschrift f. physiol. Chem.,
Band 8, p. 129.
The fact that deuteroproteoses are the primary products of
trypsin-digestion again emphasizes the natural adaptability of this
ferment to the part it has to play in the digestive process. Its
natural function is to take up the work where left by pepsin, and
carry it forward to the necessary point; and hence, when acting
upon a native proteid the primary products of its action correspond
to the secondary products of pepsin-proteolysis. Trypsin is thus
equally efficient in the digestion of all native proteids, but the
products of such action are always deuteroproteoses, peptones, and
crystalline amido-acids. It is to be remembered, however, that in
trypsin-proteolysis the deuteroproteoses and the amphopeptones must
necessarily be represented by bodies in which there is a preponderance
of anti-groups. In pepsin-proteolysis, as we have seen, the hemi- and
anti-groups of the proteid molecule remain more or less united, but in
pancreatic digestion, the formation of amphopeptone is quickly followed
by the breaking down of a portion of the hemipeptone into leucin,
tyrosin, etc. thus leaving a larger proportion of the anti-moiety in
the remaining amphopeptone.
Theoretically, at least, in the-case of a vigorous and long-continued
pancreatic digestion, all of the hemipeptone formed from any native
proteid can be converted into crystalline and other products, thus
leaving a true antipeptone resistant to the further action of trypsin.
Hence, we are prone to speak of the peptone of pancreatic digestion
as antipeptone, although, as can be readily seen, the exact nature of
the peptone, _i. e._, the relative proportion of hemi- and anti-groups
it contains, will obviously depend upon the length of the digestion
and the strength of the ferment. Again, it is possible, as certain
facts seem to suggest, that the amido-acids which are so readily
formed from hemipeptone may come in part directly from the hydration
of a portion of the hemideuteroproteose, without passing through the
preliminary stage of hemipeptone. If so, we have another source of
variation in the relative proportion of hemi- and anti-moieties in the
deuteroproteoses and peptones of pancreatic digestion. Still again, it
is to be remembered that in normal digestive proteolysis, as it occurs
in the living intestinal tract, the proteid matter to be acted upon
has already passed through certain preliminary stages in, its transit
through the stomach, as a result of which still further variations in
the proportion of hemi- and anti-groups may be possible.
It is thus plainly evident, in view of the ready cleavage of
the hemi-group into amido-acids, that the primary products of
trypsin-proteolysis, the proteoses and peptones, must necessarily be
composed in great part of those complex and semi-resistant atoms which
we include under the head of the anti-group. However much one may be
skeptical about the real existence of so-called hemi- and anti-groups,
there is no gainsaying the fact that a given weight of native proteid,
like egg-albumin or blood-fibrin, cannot be converted wholly into
crystalline or other simple products by trypsin; indeed, it is quite
significant that at the end of a long-continued treatment with an
alkaline solution of the pancreatic ferment, there is usually found
about fifty per cent, of peptone, while the other fifty per cent. of
the proteid is represented mainly by more soluble products, such as
the amido-acids. It is also significant that the peptone obtained from
an artificial pancreatic digestion, where the proteolytic action has
been long-continued and vigorous, resists the further action of the
ferment. In other words, it is the so-called antipeptone. In line with
this result is the fact that the peptones formed in pepsin-proteolysis,
when treated with an alkaline solution of trypsin, are converted into
amido-acids and other bodies of simple constitution to the extent of
about fifty per cent. This is easily explainable on the ground that
the hemi-portions of the above peptones are broken down into simple
products, while the anti-portions remain unchanged, being resistant to
the ferment and thus leading to a separation of the two groups, or at
least to the isolation of the anti-molecules.
There is much that might be cited in further support of these views,
but doubtless I have said enough to make it plainly evident that in
the pancreatic digestion of any native proteid, not more than one-half
can at the most be transformed into crystalline products, while the
other half will be represented mainly by a peptone incapable of further
change by trypsin. Similarly, the products of pepsin-proteolysis
exposed to the action of trypsin may undergo a like separation, the
hemi-groups only breaking down into simple products. Hence, the whole
theory of the hemi- and anti-moieties of the proteid molecule means
simply that of the many complex atoms composing the molecule, one-half
are easily decomposable by the pancreatic ferment, while the other half
are more resistant and make up the so-called anti-group.
In any active pancreatic digestion of either a native proteid, or of
the products of pepsin-proteolysis, the anti-group is represented
mainly by antipeptone, although there is often found a small amount of
a peculiar antialbumid-like body, insoluble in the weak alkaline fluid.
Antipeptones, thus far studied, when entirely free from proteoses, are
characterized by a low content of carbon, like the amphopeptones from
pepsin-proteolysis. The following table shows the composition of a few
typical examples:
COMPOSITION OF ANTIPEPTONES.
From From From
blood- blood- anti- From From
fibrin. fibrin. albumose. casein. myosin.
[172] [173] [174] [175] [176]
C 47.30 49.59 48.94 49.94 49.26
H 6.73 6.92 6.65 6.51 6.87
N 16.83 15.79 15.89 16.30 16.62
S 0.73 --- --- 0.68 1.16
O 28.41 --- --- 26.57 26.09
[172] Kühne and Chittenden: Studies in Physiol. Chem. Yale Univer.,
vol. ii., p. 40.
[173] J. Otto: Zeitschr. f. physiol. Chem., Band 8, p. 146.
[174] Kühne and Chittenden: Zeitschr. f. Biol., Band 19, p. 196.
[175] Chittenden: Studies in Physiol. Chem. Yale Univer., vol. iii.,
p. 101.
[176] Chittenden and Goodwin: Journal of Physiol., vol. xii., p. 34.
From these data it is evident that, while each individual peptone may
have a composition peculiar to itself, they are all alike in possessing
a relatively low content of carbon. The antialbumid, however, split off
in these hydrolytic changes, like the antialbumid formed by the action
of dilute acids at 100° C., is characterized by a correspondingly high
content of carbon and a low content of nitrogen. As an illustration,
may be mentioned the myosin-antialbumid formed in the digestion of
myosin from muscle-tissue by an alkaline trypsin-solution. This body
contains 57.48 per cent. of carbon, 7.67 per cent. of hydrogen, 13.94
per cent. of nitrogen, 1.32 per cent. of sulphur, and 19.59 per
cent. of oxygen.[177] It is only necessary to compare these figures
with those expressive of the composition of myosin-antipeptone, to
appreciate how wide a gap there is between these two products of
trypsin-proteolysis, and both members of the anti-group. Antialbumid,
however, is a peculiar product, one which is liable to crop out
somewhat unexpectedly, and with varying shades of resistance toward
the proteolytic ferments. As formed in pepsin-proteolysis, it is more
or less readily soluble in sodium carbonate, and in part readily
convertible into antipeptone by trypsin. Still, the same substance, or
at least a closely related body, makes its appearance in the form of
an insoluble residue whenever a native proteid is digested by trypsin.
At times, the amount of this insoluble product may be quite large,
even reaching to one-fourth of the total proteid matter;[178] but when
so formed in the intestine it must entail a heavy loss of nutriment,
for whenever the anti-group is split off after this fashion it becomes
very resistant to the further action of the ferment. Separating in
this manner from an artificial digestive mixture, it may be dissolved
in dilute caustic alkali, reprecipitated by neutralization, and then
once again brought into solution with dilute sodium carbonate. In this
form, it will yield some antipeptone by the further action of trypsin,
although even then a large amount of the antialbumid is prone to
separate out as a gelatinous coagulum, more or less resistant to the
further action of the ferment.
[177] Chittenden and Goodwin: Journal of Physiol., vol. xii., p. 36.
[178] Kühne und Chittenden: Ueber die nächsten Spaltungsproducte der
Eiweisskörper. Zeitschr. f. Biol., Band 19, p. 196.
The peculiar action of trypsin, however, as a proteolytic enzyme is
shown in the production of a row of crystalline nitrogenous bodies
of simple constitution whenever the ferment is allowed to continue
its action for any length of time, either on native proteids or on
proteolytic products containing the hemi-group. This, to be sure, is a
fact long known, but it gains added significance as year by year new
bodies are discovered as products of trypsin-proteolysis with various
forms of proteid matter. The very character of the bodies originating
in this manner gives evidence of the far-reaching decompositions
involved; decompositions which are perhaps attributable as much to the
innate tendencies of the proteid material as to the specific action of
the ferment. As representatives of this peculiar line of cleavage, we
have first the well-known bodies, leucin and tyrosin; leucin, a body
belonging to the fatty acid series, long known as amido-caproic
acid, but now generally considered as amido-isobutylacetic acid,
(CH_{3})_{2} CHCH_{2} CH(NH_{2}) COOH; and tyrosin, a body
belonging to the aromatic group, having the formula
OH
╱
C_{6}H_{4}⧼
╲
CH_{2} CH(NH_{2}) COOH,
and known as oxyphenyl-amido-propionic acid.
These two bodies are therefore representatives of two distinct groups
or radicals present in the hemi-portion of the proteid molecule; the
first belonging to the fatty acid series, the second to the aromatic
group from which come such well-known bodies as indol, skatol, benzoic
acid, and other substances prominent in proteid metabolism. Moreover,
these two hydrolytic products of trypsin-proteolysis are formed in
considerable quantity, at least in an artificial digestion. Thus,
Kühne has reported the finding of 9.1 per cent. of leucin and 3.8 per
cent. of tyrosin as the result of a typical digestion, and I have
tried many similar experiments with like results. Further, we know
from observations made by different investigators that both leucin and
tyrosin may be formed in considerable quantities in trypsin-proteolysis
as it occurs in the living intestine. But to this point we shall return
later on.
Besides leucin and tyrosin, aspartic acid and glutamic acid have long
been known as decomposition-products of the vegetable proteids. Thus,
both acids were discovered by Ritthausen and Kreusler[179] in the
cleavage of such proteids by boiling dilute acid. Hlasiwetz and
Habermann[180] likewise obtained aspartic acid in large quantity by
the breaking down of animal proteids under the influence of bromine.
Further, Siegfried[181] has recently obtained glutamic acid as a
product of the decomposition of the phosphorus-containing proteid,
reticulin, from adenoid tissue. As products of trypsin-proteolysis,
Salkowski and Radziejewski[182] found aspartic acid in the digestion
of blood-fibrin; and v. Knieriem[183] likewise obtained it in the
digestion of gluten from wheat. Both of these acids belong to the
fatty acid series, the aspartic acid being a dibasic acid,
COOH. CH_{2}CH(NH_{2}). COOH, or amido-succinic acid, while glutamic
acid, COOH. C_{3}H_{5}(NH_{2}). COOH, is likewise a dibasic acid,
known as amido-pyrotartaric acid.
[179] Verbreitung der Asparaginsäure und Glutaminsäure unter den
Zersetzungs-producten der Proteinstoffe. Journal f. prakt. Chemie, Band
3, p. 314.
[180] Ueber die Proteinstoffe. Liebig’s Annalen, Band 159, p. 304.
[181] Ueber die chemischen Eigenschaften des Reticulirten Gewebes.
Habilitationschrift. Leipzig, 1892.
[182] Bildung von Asparaginsäure bei der Pancreas-Verdauung. Bericht.
d. Deutsch. chem. Gesellsch., Band 7, p. 1050.
[183] Asparaginsäure, ein Product der künstlichen Verdauung von Kleber
durch die Pancreas-Drüse. Zeitschr. f. Biol., Band 11, p. 198.
Of more interest physiologically, are the recently discovered
nitrogenous bases lysin and lysatinin, or lysatin. These two bodies
were first identified by Drechsel[184] and his co-workers as products
of the decomposition of various proteids, when the latter are boiled
with hydrochloric acid and stannous chloride. They were first obtained
by Drechsel as cleavage products of casein.[185] Later, Ernst
Fischer,[186] working under Drechsel’s direction, separated them as
decomposition-products of gelatin; while Siegfried[187] obtained them
as products of the cleavage of conglutin, gluten-fibrin, hemiprotein,
and egg-albumin, by boiling with hydrochloric acid and stannous
chloride. In all of these cases it is obvious, from the method of
treatment pursued, that the two bodies result from a simple hydrolytic
cleavage of the proteid molecule. Hence, it might be assumed that
these two bases would likewise be formed in trypsin-proteolysis. This
assumption, Hedin,[188] working in Drechsel’s laboratory, has proved to
be correct, and furthermore he has shown that the amount of these bases
formed in pancreatic digestion is not inconsiderable. Thus, as products
of the digestion of three kilos. of moist blood-fibrin with an alkaline
solution of trypsin, 28 grammes of pure platino-chloride of lysin were
obtained, and sufficient lysatinin to establish its identity.
[184] Der Abbau der Eiweissstoffe. Du Bois-Reymond’s Archiv. f.
Physiol., p. 248. 1891.
[185] Zur Kenntniss der Spaltungsproducte des Caseins. _Ibid._, p. 254.
1891.
[186] Ueber neue Spaltungsproducte des Leimes. _Ibid._, p. 265. 1891.
[187] Zur Kenntniss der Spaltungsproducte der Eiweisskörper. _Ibid._,
p. 270. 1891.
[188] Zur Kenntniss der Producte der tryptischen Verdauung des Fibrins.
_Ibid._, p. 273. 1891.
Lysin has the composition of C_{6} H_{14} N_{2} O_{2}, being a
diamido-caproic acid, a homologue of diamido-valerianic acid. Hence,
this body, like leucin or amido-caproic acid, is a representative of
the fatty acid group, the chemical relationship between the two bodies
being plainly apparent from their constitution. The constitution
of lysatinin is less definitely settled, but apparently it has the
composition of a creatin, its formula being C_{6}H_{13}N_{3}O_{2}, in
which case it might be more appropriately termed lysatin. The special
point of interest, however, connected with this latter body as a
product of trypsin-proteolysis is the fact that by simple hydrolytic
decomposition, all chance of oxidation being excluded, it can break
down into urea.[189] For years, chemists have been seeking to trace
out the line of cleavage or decomposition by which urea results in
proteid metabolism. In the nutritional changes of the body, nearly all
the nitrogen of the ingested proteid food is excreted in the form of
urea, but chemists working with dead food-albumin have been heretofore
unable to break down proteid matter directly into urea. This, however,
Drechsel has now succeeded in doing, and it is to be especially noted
that the line of decomposition or cleavage is simply one of hydration,
in which the proteid molecule, either through the action of boiling
dilute acids, or through the more subtle influence of the hydrolytic
enzyme, trypsin, is gradually broken down into cleavage products, from
one or more of which comes lysatin. The very resemblance of this body
to creatin suggested that, since the latter breaks down into urea and
sarcosin when boiled with baryta water, lysatin might possibly behave
in a similar manner. This, as has been previously stated, was found to
be the case, and Drechsel obtained from ten grammes of a double salt of
lysatin and silver one gramme of urea nitrate, by simple boiling with
baryta water.
[189] Drechsel: Ueber die Bildung von Harnstoff aus Eiweiss. Du
Bois-Reymond’s Archiv f. Physiol., p. 261. 1891.
It is thus evident that a certain amount of urea may come from the more
or less direct hydrolysis of proteid matter in the intestinal canal,
all but the last steps in the process being the result of the ordinary
cleavage processes incidental to trypsin-proteolysis. This fact affords
additional evidence of the profound changes set in motion by this
proteolytic enzyme. It is not, of course, to be understood that all the
urea formed in the body has its origin in this manner. Such a method of
decomposition taking place in the intestinal tract would be exceedingly
unphysiological and wasteful, but we can readily see how such a line of
cleavage might result in inestimable gain to the economy in cases where
excess of proteid food has been ingested. Under such circumstances, a
portion of the surplus might be broken down directly in the intestine
into this urea-antecedent, and thus quickly removed from the system
with a minimum amount of effort on the part of the economy. Drechsel
estimates that about one-ninth of the urea daily excreted may come from
the direct decomposition of lysatin, the latter obviously having its
origin in trypsin-proteolysis.
Another product of trypsin-proteolysis which has long been recognized,
although its real nature has not been known, is tryptophan or
proteinochromogen. This body is not only a product of the pancreatic
digestion of proteids, but it is also formed whenever native proteids
are broken down through any influence whatever, the substance coming
presumably from the hemi-moiety of the molecule. It is especially
characterized by the bright-colored compound it forms with either
chlorine or bromine, so that for a long time it went by the mystical
name of the “bromine body.” When brought in contact with either of
these agents, it immediately combines with them to form a new compound
of an intense violet color, termed proteinochrome. This constitutes
the usual test for its presence, a little bromine water, for example,
quickly bringing out a violet color when added to a fluid containing
the chromogen. The body is readily soluble in alcohol, and hence can
be easily separated from the primary products of trypsin-proteolysis,
such as the proteoses and peptones. Krukenberg considered the substance
not a true proteid, but rather a body belonging to the indigo-group;
but Stadelmann, who has given the matter a very thorough investigation,
comes to the conclusion that it is truly a proteid body, in part
closely related to peptone, although in many ways quite different.
The following composition of bromine proteinochrome, as determined by
Stadelmann,[190] shows the general nature of the compound formed when
bromine combines with the chromogen:
_A_ _D_
C 49.00 48.12
H 5.28 5.09
N 10.99 11.92
S 3.77 3.10
O 11.01 12.00
Br 19.95 19.77
[190] Ueber das beim tiefen Zerfall der Eiweisskörper entstehende
Proteinochromogen, den die Bromreaction gebenden Körper. Zeitschr. f.
Biol., Band 26, p. 521.