detection of so-called free and combined hydrochloric acid in the
stomach-contents, or the special significance of such findings in
health and disease. I cannot refrain, however, in connection with what
has been said above concerning the proteolytic action of pepsin in the
presence of combined acid, from saying a word concerning the usual
deductions drawn from the absence of free acid in the stomach-contents.
As Langermann[112] has recently expressed it, we have methods for
discriminating between free and combined acid; we can, moreover,
determine the amount of free acid, but is it not equally important to
be able to say something definite concerning the amount of combined
acid in the stomach-contents? Even in the absence of free hydrochloric
acid there may be a sufficient amount of HCl secreted to answer all the
purposes of digestion, and yet at no time may there be any free acid
present to be detected by the various color-tests ordinarily made use
of. I am aware that in ordinary examinations of the stomach-contents
after a test meal the results are essentially comparative, and possibly
all that are necessary for clinical purposes. What I wish to emphasize,
however, is that in order to pass conclusively upon tsufficiency or
insufficiency of the gastric secretion, it is wise to know not only the
total acidity of the stomach contents and whether there is free acid
or not, but to know more about the amount of combined acid present.
Thus, there is a natural tendency to divide the fluids withdrawn from
the stomach into three groups, viz., those which contain free acid in
moderate amount, those which contain free acid in excess, and those
in which free acid is entirely absent; but in the latter group, there
may be very marked differences in the amount of acid combined with
the proteid and other material present. It appears to me that one of
the questions to be answered is whether there is sufficient combined
HCl present to meet all the requirements for digestion. If there
is, that gastric juice may be just as normal as the one containing
free mineral acid, and yet, according to our present tendencies,
we should be inclined to call the juice containing no free acid
abnormal, although there may be sufficient combined acid present to
meet all the requirements for digestion. Hence, in examination of the
stomach-contents, it is well to consider the use of those methods
which tend to throw light upon the amount of combined acid present,
for in my opinion it is only by a determination of the total amount of
combined acid that we can arrive at a true estimate of the extent of
the HCl deficiency. Obviously, in simple clinical examinations of the
stomach-contents after a test meal, where proteid matter is not present
in large amount, free acid may reasonably be expected to appear after
a definite period; but in any event, it is well to remember that free
hydrochloric acid is not absolutely indispensable for fairly vigorous
proteolytic action, and that in the presence of moderate amounts of
proteid matter a large quantity of acid is required to even saturate
the albuminous material.
[112] Virchow’s Archiv, Band 128, p. 408.
Consider for a moment the amount of acid a given weight of proteid
will combine with, before a reaction for free acid can be obtained.
Thus, Blum[113] has stated that 100 grammes of dry fibrin will require
9.1 litres of 0.1 per cent. hydrochloric acid to completely saturate
it. Hence, with a daily consumption of 100 grammes of proteid, there
would be needed for gastric digestion 4.5 litres of 0.2 per cent.
hydrochloric acid daily, and even this would not suffice to give any
free acid, assuming that none of the acid is used over again. The
results I have already given for egg-albumin tend to show that 1
gramme of pure albumin, free from inorganic salts, when dissolved in
a moderate amount of water will combine with about 30 c.c. of 0.2 per
cent. hydrochloric acid. Consequently, on this basis, 100 grammes of
dry egg-albumin will combine with 3 litres of 0.2 per cent. HCl, and
not until this amount of acid has been added to such a mixture will
reaction for free acid be obtained with Günzburg’s reagent. Hence
we can easily see, in view of these figures, that the production of
hydrochloric acid by the gastric glands may at times be very extensive,
without the stomach-contents necessarily containing free acid.
[113] Zeitschr. f. klin. Medicin, Band 21, p. 558.
While I am by no means willing to agree with Bunge[114] that the
chief importance of the acid of the gastric juice is its action as
an antiseptic, I am decidedly of the opinion that the lack of free
hydrochloric acid in the stomach-contents is more liable to cause
disturbance through the consequent unchecked development of bacteria
than through lack of proteolytic action, assuming, of course, the
presence of a reasonable amount of combined HCl. The hydrochloric
acid of the gastric juice unquestionably plays a very important part
in checking the growth and development of many pathogenic bacteria,
as well as of less poisonous organisms, which are taken into the
mouth with the food. On all, or at least on nearly all of these
organisms, hydrochloric acid exerts a far greater destructive action
when free than when combined with proteid matter. As Cohn[115] has
plainly shown, both hydrochloric acid and pepsin-hydrochloric acid
quickly hinder acetic- and lactic-acid fermentation, but when the
acid is combined with peptone, for example, it is no longer able to
exercise the same inhibitory influence. It is also important to note
that the lactic-acid ferment is not so sensitive to hydrochloric
acid as the acetic-acid ferment. Consequently, when lactic-acid
fermentation is once developed a comparatively large amount of HCl is
required to arrest it. Hence, as we all know, a diminished secretion
of hydrochloric acid renders possible acid fermentation of the
stomach-contents, as well as putrefactive changes which would not occur
in the presence of free HCl, and which are very incompletely checked
when the acid is over-saturated with proteid matter.
[114] Physiologische und Pathologische Chemie, p. 153.
[115] Ueber die Einwirkung des künstlichen Magensaftes auf
Essigsäure-und Milchsäure gährung. Zeitschr. f. physiol. Chemie, Band
14, p. 75. See also Hirschfeld: Pflüger’s Archiv f. Physiol., Band 47,
p. 510.
Pepsin-proteolysis, however, may proceed, to some extent, at least,
even though a small amount only of combined acid is present. The
combined acid, however, must be hydrochloric acid, if proteolysis is to
be at all marked. To be sure, pepsin will act in the presence of lactic
acid, as well as in the presence of other organic acids, and inorganic
acids, likewise, but such action at the best is considerably weaker
than the action of pepsin-hydrochloric acid.[116]
[116] Chittenden and Allen: Influence of Various Inorganic and
Alkaloidal Salts on the Proteolytic Action of Pepsin-Hydrochloric Acid.
Studies in Physiol. Chem., Yale University, vol. 1, pp. 91, 94.
The ferment pepsin can exert its _maximum_ action only in the
presence of free hydrochloric acid. There must be sufficient HCl to
combine with all of the proteid matter present, and the products of
proteolysis as fast as they are formed, if digestion is to be rapid
and attended with the formation of a large proportion of the final
products of proteolysis. It is under such conditions that our study
of pepsin-proteolysis is usually conducted. Further, it is to be
remembered that our knowledge of the products of such proteolytic
action depends almost entirely upon data accumulated by artificial
digestive experiments. In no other way can we be absolutely certain
of the conditions under which the proteolysis is accomplished, for it
is a significant fact, perhaps plainly evident from what has already
been said in the preceding lecture, that the character of the products
resulting from ordinary proteolysis is dependent in great part upon
the attendant circumstances. Thus, with a relatively small amount of
acid, and perhaps also of pepsin, the initial products of proteolysis
are especially prominent, while with an abundance of both pepsin and
free acid, coupled with long-continued action, the final products
predominate. Between these two extremes there are many possible
variations, as was, I think, made clear in the previous lecture. At
the same time, it is to be noticed that these differences are mainly
differences in the _proportion_ of the several products, rather than in
the nature of the resultant bodies.
In a general way, the products of pepsin-proteolysis may be divided
into three main groups, viz., bodies precipitated by neutralization and
represented mainly by the so-called syntonin or acid-albumin; bodies
precipitated by saturation of the neutralized fluid with ammonium
sulphate and represented by proteoses; bodies non-precipitable by
saturation with ammonium sulphate and represented by amphopeptones. The
relationship of the individual products may be clearly seen from the
following scheme, arranged after the plan suggested by Neumeister.
NATIVE PROTEID.
|
|
Syntonin.
╱ ╲
╱ ╲
╱ ╲
Protoproteose. Heteroproteose.
| (dysproteose).
| |
Deuteroproteose. Deuteroproteose.
| |
Peptone. Peptone.
It is, of course, to be understood that this is not intended to
represent anything more than the order of formation of the several
bodies, no attention being paid here to the hemi- or anti-character
of the several products, or classes of products. Thus, proto and
heteroproteose are primary bodies formed directly from the initial
product syntonin by the further action of the ferment. In the same
sense, deuteroproteose is a secondary proteose, being formed by the
further hydration of the primary body. Lastly, peptones, the final
products of pepsin-proteolysis, are the result of the hydration and
possible cleavage of deuteroproteoses. Further, in almost every gastric
digestion there is also formed a small amount of antialbumid, a product
insoluble in dilute hydrochloric acid and which consequently appears
as an insoluble residue. This body is very resistant to the action of
pepsin-acid when once formed, but may be slowly converted, in part at
least, into a soluble antialbumose and thence into antipeptone.
All of these bodies can be readily identified in any digestive
mixture containing them by a few simple reactions. Thus, after having
removed any acid-albumin or syntonin present by neutralization, the
concentrated fluid can be tested at once. If primary proteoses are
present, the neutral fluid will yield a more or less heavy precipitate
on addition of crystals of rock-salt, precipitation being complete
only when the fluid is saturated with the salt. Further, if the
proteoses are present in not too small quantity, nitric acid added drop
by drop to the neutralized fluid will produce a white precipitate,
readily soluble on application of heat but reappearing as the solution
cools. If primary proteoses are wholly wanting, then no precipitate
will be obtained by acid unless the fluid is saturated with salt, in
which case a portion of the deuteroproteose will be precipitated.
The two primary proteoses differ from each other especially in
solubility; protoproteose being readily soluble in water alone, while
heteroproteose is soluble only in salt solutions, dilute acids, and
alkalies. Hence, when these two bodies are precipitated together by
saturation with salt, they may be readily separated by dissolving them
in a little dilute salt solution, and dialyzing the fluid in running
water until the salt is entirely removed; heteroproteose will then be
precipitated, while the proto-body remains in solution.
By long contact with water, and even with concentrated salt solutions,
heteroproteose tends to undergo change into a semi-coagulated form,
named dysproteose, insoluble in dilute sodium-chloride solutions.
This body can be reconverted into heteroproteose, in part at least,
by solution in dilute acid, or alkali, and reprecipitation by
neutralization.
As a class, the proteoses are characterized by far readier
solubility in water than native proteids, by a far greater degree of
diffusibility, by non-coagulability by heat and by alcohol, although
precipitable by the latter agent. Further, nearly all proteose
precipitates are exceedingly sensitive toward heat, tending to dissolve
as the fluid is warmed and reappearing as the solution cools. In
fact, this peculiarity often serves as a means of identification.
Potassium ferrocyanide and acetic acid, picric acid in excess, and
likewise cupric sulphate, all precipitate the primary proteoses, while
deuteroproteose is only slightly affected by these reagents, or indeed
not at all.
In order to separate the secondary proteose from the primary bodies
in the absence of peptones, the fluid is neutralized as nearly as
possible, and then, after suitable concentration, is saturated with
sodium chloride for the partial precipitation of the primary proteoses.
To the clear filtrate, acetic acid[117] is added drop by drop as long
as a precipitate results, the latter being composed of a mixture of
protoproteose and deuteroproteose. That is to say, protoproteoses
are not completely precipitated from neutral solutions by saturation
with salt alone; a little acid is required to complete it, but this
tends to bring down a certain amount of deuteroproteose. From this
filtrate, however, the deutero-body can be separated in a pure form by
dialyzing away the salt and acid, and then concentrating the fluid and
precipitating with alcohol. When the proteoses are mixed with peptones,
the former must first be separated collectively by saturation of the
fluid with ammonium sulphate.
[117] Saturated with sodium chloride.
Peptones are especially characterized by non-precipitation with the
ordinary precipitants for proteid bodies, and especially by the
fact that they are wholly indifferent to saturation with ammonium
sulphate either in neutral, acid, or alkaline fluids. This reaction,
which constitutes the main, and perhaps the only absolute method
of separating peptones from proteoses must be carried out with
great thoroughness in order to insure a complete precipitation of
deuteroproteose. The latter stands midway between primary proteoses and
peptones in many respects, and seems to share with peptones something
of a tendency to resist precipitation by the ammonium salt. Indeed, as
Kühne[118] has recently pointed out, the last traces of deuteroproteose
can be precipitated from the fluid only by long continued boiling of
the ammonium sulphate-saturated fluid, and even then it is seldom
complete unless the reaction of the fluid is alternately made neutral,
acid, and alkaline, and the heating continued for some time after each
change in reaction. Under such circumstances, the last portions of
deuteroproteose separate from the salt-saturated fluid and float on the
surface in the form of an oily or gummy mass, while the true peptone
remains in the fluid absolutely non-precipitable by the salt.
[118] Erfahrungen über Albumosen und Peptone. Zeitschr. f. Biol., Band
29, p. 2.
In this filtrate, peptone can be detected by adding to a small portion
of the fluid a very large excess of a strong solution of potassium
hydroxide, followed by the addition of a few drops of a very dilute
solution of cupric sulphate. If peptone is present a bright red color
will appear, the intensity of which, with the proper amount of cupric
sulphate, will be proportional to the amount of peptone present. If it
is desired to separate the peptone from the ammonium-sulphate-saturated
fluid, there are several methods available, of which the following is
perhaps the most satisfactory: The fluid is concentrated somewhat,
and set aside in a cool place for crystallization of a portion of the
ammonium salt. The fluid is then mixed with about one-fifth its volume
of alcohol, and allowed to stand for some time, when it separates
into two layers--an upper one, rich in alcohol, and a lower one, rich
in salts. The latter is again treated with alcohol, by which another
separation of the same order is accomplished. Finally, the lighter
alcoholic layers containing the peptone are united, and exposed to a
low temperature until considerable of the contained salt crystallizes
out. The fluid is then concentrated, and after addition of a little
water is boiled with barium carbonate until the fluid is entirely
free from ammonium sulphate. Any excess of baryta in the filtrate is
removed by cautious addition of dilute sulphuric acid, after which the
concentrated fluid, reduced almost to a sirupy mass, is poured into
absolute alcohol for precipitation of the peptone.
So separated, the peptone formed in gastric digestion is exceedingly
gummy, but can be transformed into a yellowish powder, very
hygroscopic, of more or less bitter taste, and, when thoroughly dry,
dissolving in water with a hissing sound and with considerable
development of heat, like phosphoric anhydride.[119]
[119] Kühne and Chittenden: Peptones. Studies in Physiol. Chem., Yale
University, vol. 2, p. 14.
I have introduced these dry chemical facts, none of which are
especially new, because I deem them of considerable importance and
because they are not very generally known. In fact, there seems to be
a tendency on the part of some who are more or less familiar with the
advances made in our knowledge of the products of pepsin-proteolysis
to question the existence of these different bodies, or to show at
least a spirit of indifference toward these recent facts which have
been gradually accumulated, and I may say accumulated at the expense
of considerable labor. The time is past for calling the products of
gastric digestion peptones; it is time for a full recognition of the
fact that pepsin-proteolysis is synonymous with the production of a row
of bodies, chemically and physiologically distinct from each other,
each endowed with individuality enough to admit of certain detection,
and all bearing a certain specific and harmonious relationship to their
neighbors, the other members of the series.
Further, it is not enough to admit the formation of a single
intermediate body, midway between syntonin and peptone. The so-called
propeptone of the past is simply a mixture of proteoses, of ever
changing composition, varying with each change in the proportion of the
component proteoses. Each of these proteoses can be detected, under
suitable conditions, in the products of every artificial digestion
as well as in the stomach-contents, and no better measure of the
proteolytic power of the natural stomach-secretion can be devised
than a study of the character of the individual bodies present in the
stomach-contents after a suitable test meal. The proper tests and
separations can be made with a small amount of the filtered fluid, and
much light thrown upon the digestive power of the secretion by even a
rough estimate of the proportion of primary and secondary proteoses
and peptones formed in a given time, after the ingestion of a certain
amount of proteid food.
In pepsin-proteolysis we have to deal, in my opinion, with a series of
progressive hydrolytic changes in which peptones are the final products
of the transformation. Commencing with the formation of acid-albumin
or syntonin, hydrolysis and cleavage proceed hand in hand, under the
guiding influence of the proteolytic enzyme, and each onward step in
the process is marked by the appearance of a new body corresponding to
the extent of the hydrolysis; each body, perhaps, being represented
by a row or series of isomers, all externally alike, but different
in their inner structure, according to the proportion of hemi- and
anti-groups contained in the molecule. As opposed to this theory, we
have the older views of Maly,[120] Herth,[121] Henninger[122] and
others, based upon observations which tend to show that peptones do
not differ in chemical composition from the proteids which yield them.
As a matter of fact, the products then analyzed were not peptones at
all; they were merely the _primary_ products of pepsin-proteolysis,
_i. e._, what we now term primary proteoses, and it is time we stopped
using such data to enforce the theory that peptones are polymers of the
proteids from which they are derived.
[120] Ueber die chemische Zusammensetzung und physiologische Bedeutung
der Peptone. Pflüger’s Archiv f. Physiol., Band 9, p. 585.
[121] Ueber die chemische Natur des Peptones und sein Verhältniss zum
Eiweiss. Zeitschr. f. physiol. chem., Band 1, p. 277.
[122] De la Nature et du rôle physiologique des peptones. Paris, 1878.
In 1886, the writer, in conjunction with Professor Kühne, commenced
a study of the various cleavage products[123] formed by the action
of pepsin-hydrochloric acid from the better characterized and
purer proteids, this being a continuation of our earlier work on
the proteoses and peptones formed from blood-fibrin, serum-albumin,
etc. This work I have continued in my laboratory up to the present
time, with many co-workers, and as a result we have to-day a series
of observations gradually accumulated during these last seven years,
some the results of work carried on this last year, which speak in
no uncertain way of the character of both the primary and secondary
products of pepsin-proteolysis. Furthermore, in attempting to settle
this question once for all, I have selected for study examples from
the various classes of both animal and vegetable proteids; and as
representatives of the latter have had carried out two lengthy series
of experiments on the crystallized proteids which occur so abundantly
in some seeds, on the assumption that these crystalline bodies would
furnish a certain guarantee of purity which might naturally be lacking
in the amorphous proteids of animal origin. Some of these results are
now placed together in the following tables, a study of which reveals
some very interesting facts:
[123] Globulin and Globuloses. Studies in Physiol. Chem. Yale
University, vol. ii., p. 1.
COMPOSITION OF PROTEOLYTIC PRODUCTS FORMED BY PEPSIN-HYDROCHLORIC ACID.
_Proteolysis of Blood-fibrin._
=====================================================
| Mother | Proto- | Hetero- | Deutero- | Ampho-
|Proteid.|fibrinose.|fibrinose.|fibrinose.|peptone.
| | [124] | [124] | [124] | [125]
--+--------+----------+----------+----------+--------
C | 52.68 | 51.50 | 50.74 | 50.47 | 48.75
H | 6.83 | 6.80 | 6.72 | 6.81 | 7.21
N | 16.91 | 17.13 | 17.14 | 17.20 | 16.26
S | 1.10 | 0.94 | 1.16 | 0.87 | 0.77
O | 22.48 | 23.63 | 24.24 | 24.65 | 27.01
--+--------+----------+----------+----------+--------
[124] Kühne and Chittenden: Zeitschr. f. Biol., Band 20, p. 40.
[125] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer.,
vol. ii., p. 40.
_Proteolysis of Paraglobulin._[126]
============================================
| Mother | Proto- | Hetero- | Deutero-
|Proteid.|globulose.|globulose.|globulose.
--+--------+----------+----------+----------
C | 52.71 | 51.57 | 52.10 | 51.52
H | 7.01 | 6.98 | 6.98 | 6.95
N | 15.85 | 16.09 | 16.08 | 15.94
S | 1.11 }| | |
O | 23.24 }| 25.36 | 24.84 | 25.59
--+--------+----------+----------+----------
[126] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer.,
vol. ii, p. 12.
_Proteolysis of Coagulated Egg-albumin._
==================================================
| Mother | Proto- | Hetero- |Deutero- | Hemi-
|Proteid.|albumose.|albumose.|albumose.|peptone.
| | [127] | [127] | [127] | [128]
--+--------+---------+---------+---------+--------
C | 52.33 | 51.44 | 52.06 | 51.19 | 49.38
H | 6.98 | 7.10 | 6.95 | 6.94 | 6.81
N | 15.84 | 16.18 | 15.55 | 15.77 | 15.07
S | 1.81 | 2.00 | 1.63 | 2.02 | 1.10
O | 23.04 | 23.28 | 23.81 | 24.08 | 27.64
--+--------+---------+---------+---------+--------
[127] Chittenden and Bolton: _Ibid._, vol. ii, p. 153.
[128] Kühne and Chittenden: Zeitschr. f. Biol., Band 19, p. 201.
_Proteolysis of Casein from Milk._
===================================================
| Mother | Proto- |Hetero- |α Deutero-|β Deutero-
|Proteid.|caseose.|caseose.| caseose. | caseose.
| | [129] | [130] | [129] | [129]
--+--------+--------+--------+----------+----------
C | 53.30 | 54.58 | 53.88 | 52.10 | 47.72
H | 7.07 | 7.10 | 7.27 | 6.93 | 6.73
N | 15.91 | 15.80 | 15.67 | 15.51 | 15.97
S | 0.82 }| | | |
O | 22.03 }| 22.52 | 23.18 | 25.46 | 29.58
--+--------+--------+--------+----------+----------
[129] Chittenden: Studies in Physiol. Chem., Yale Univer., vol. iii.,
p. 80.
[130] Chittenden and Painter: _Ibid._, vol. ii., p. 195.
_Proteolysis of Myosin from Muscle._[131]
====================================================
|Mother Proteid.|Protomyosinose.|Deuteromyosinose.
--+---------------+---------------+-----------------
C | 52.82 | 52.43 | 50.97
H | 7.11 | 7.17 | 7.42
N | 16.77 | 16.92 | 17.00
S | 1.27 | 1.32 | 1.22
O | 21.90 | 22.16 | 23.39
--+---------------+---------------+-----------------
[131] Kühne and Chittenden: _Ibid._, vol. iii, p. 147.
_Proteolysis of Elastin._[132]
==+===============================================
|Mother Proteid.|Protoelastose.|Deuteroelastose.
--+---------------+--------------+----------------
C | 54.24 | 54.52 | 53.11
H | 7.27 | 7.01 | 7.08
N | 16.70 | 16.96 | 16.85
S}| | |
O}| 21.79 | 21.51 | 22.96
--+---------------+--------------+----------------