E. A. (Edward Albert) Sharpey-Schäfer.

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by producing molecular vibration, causes a chemical reaction to take
place with great rapidity. It is very likely that in many cases,
especially those in which the catalytic agent is merely required to
be present in traces, that there is no intermediate substance formed,
and that the catalytic agent acts in a physical manner, inducing a
compound already unstable to pass into a more stable condition. It
is not even necessary that the substance should be unstable in the
usual sense of the word, but only that the new products should be

^ A similar oxygen-carrier, of oxygen to be used in tissue metabolism, is found in
hremoglobin, which may be looked upon as a catalytic agent, taking up oxygen, parting
with it to bring about a reaction, the details of which we do not know, and so becoming
regenerated and coming out of the total process unchanged.


more stable ; or, in other words, that there should be energy set free in
the process of change.

In ferment action, the chemical energy of the resulting products
is always less than that of the substances from which they were formed ;
this is shown by the heats of combustion of the end products amounting
to less than those of the initial products.

The action of ferments is hence in all respects analogous to that of
catalytic agents ; there is a passage from a less stable to a more stable
condition, which is brought about by an agent which is not itself altered
in the process.

The two principal hypotheses are then — (1) That the enzyme
combines with the substance on which it is acting, and that the unstable
compound so formed decomposes, yielding the new substance and
regenerating the enzyme ; (2) that the enzyme is in a state of molecular
movement, which induces a molecular movement in the fermentable
substance, or increases such a movement when already present, so that
the molecule breaks up, over-swings, or over-vibrates as it were, into a
more stable condition, so giving rise to new substances.

Nature of the chemical change. — Somewhat more is known of the
nature of the chemical changes induced by the ferments than of the
mode in which they bring about such changes. It is probable that in
all cases ferment action is accompanied by hydrolysis, i.e. the taking
up of the elements of water.^ This is known with certainty to be the
case in all actions of diastatic and inverting ferments, and is very pro-
bably true also for proteolytic ferments. This subject will be considered
more in detail in treating of the specific action of the various enzymes
on the different classes of foodstuffs ; reference will only be made here
to the general arguments which go to show that such a process of
hydrolysis is a universal accompaniment of ferment action.

1. In many cases the composition of the products of the fermentation
compared with that of the initial substance shows directly a taking up
of water. In those in which this is not so, carbonic anhydride is usually
one of the constituents, and if this be considered as united with the
elements of a water molecule to form carbonic acid, as it probably is
when formed in the reaction, water is taken up here also. In all cases,
however, whether the products of the reaction directly show the taking
up of water or not, the presence of water is essential to the reaction,
for no ferment is known which will act otherwise than in the presence
of water.

2. Again, the action of any of the ferments may be closely imitated
by the action on the several fermentable or digestible materials of
dilute acids or alkalies, and these are recognised throughout the domain
of organic chemistry as the most powerful hydrolytic agents known.

3. It has been shown that in the case of coagulation by fibrin ferment
an increase of weight of dried material takes place, probably due to the
elements of water being taken up in the process. This was demonstrated
by taking two equal portions of plasma, allowing one to clot and not the
other, and then drying both under similar conditions, when the clotted
sample was found to weigh a half per cent, more than the other.^

1 Hoppe-Seyler, Arcli. f. d. ges. Physiol., Bonn, 1876, Bd. xii. S. 1 ; Nencki, Journ.
f. praJct. Chem., Leipzig, 1879, Bd. xvii. S. 105.

^ Observation by A. Schmidt, communicated by G. Tamman, Ztschr. f. physiol. Chem.,
Strassburg, 1892, Bd. xvi. S. 271.


Rate of zymolysis ^ or enzymic action.— The rate at which diges-
tion goes on in any digestive Huid varies chiefly with the following
conditions, namely — (1) The temperature, (2) the reaction, (3) the con-
centration of the products of digestion, (4) the concentration of the
digestive enzyme, (5) the condition of the material to be digested.

Temperature. — The digestive enzymes are very sensitive to changes
in temperature ; they all act most energetically at or slightly above the
body temperature. The point of greatest acti^dty is called the optimum
point ; as the temperature varies, either above or below this point, the
rapidity of action of the enzyme slackens ; and, as the interval apart from
the optimum point is increased, a point is finally reached at which the
action of the enzyme is no longer appreciable. Any temperature
markedly above that of optimum action slowly destroys the enzyme,
and this destructive action in all cases becomes very rapid at tempera-
tures varying (between 50° and 65° C.) with the particular ferment, the
reaction of the fluid in which it is so heated, and the degree of its dilu-
tion.2 On the other hand, low temperatures, though they slow and
finally stop ferment action, do not destroy the ferment ; this recovers its
activity completely when the temperature is again raised, even though
the temperature has been kept at - 5° C. for several hours.^

Reaction. — The variation in chemical reaction of the fiuid in which
they act has a similar effect on enzymes to that of variation in tempera-
ture. For each of the digestive enzymes there is a particular reaction,
and degree of that reaction, at which it acts with maximum power. A
departure from this degree of acidity or alkalinity causes a more or less
rapid diminution in the speed with which the enzyme acts, and a
sufficient amount of departure from the optimum reaction causes the
destruction of the enzyme. Some of the enzymes act in solutions of
either acid, neutral, or alkaline reaction, provided always that the
reaction does not stray too widely from that at which they act best ;
examples of such are ptyalin and trypsin. Others only act with one
specific reaction, and are rapidly destroyed if the reaction changes from
this. Examples of these are pepsin, only active in acid solution, and
rapidly destroyed by a trace of alkalinity ; and the fat-splitting ferment
of the pancreas, active only in alkaline or neutral solutions, and rapidly
destroyed by acid.

Accumulation of dissolved products of action. — Accumulation of the
products of the action of an enzyme in the solution acts unfavourably
upon its continued action, slowing and finally altogether checking it.*
This action may be to some extent prevented by removing the products
formed by dialysis, or diluting them by the addition of water. In the
latter case, however, the ferment is also diluted, and in the former, since
the products of digestion in most cases have no very high diffusive
power, the removal is very slow and incomplete.

Eemoval of the digestive products by dialysis has, in addition, the
disadvantage that the digestive solution is diluted by the osmosis, due to

1 This term is that proposed by Sheridan Lea, Journ. Physiol., Cambridge and London,
1890, voL xi. p. 254.

- V. Wittich, Arch.f. d. ges. Physiol., Bonn, 1869, Bd. ii. S. 193 ; 1870, Bd. iii. S. 339.

^ Bidder u. Schmidt, " Die Verdauungssiifte, etc."

* Brucke, Sitzungsb. d. k. Akad. d. JFissensch., Wien, 1862, Bd. xliii. S. 601 ;
"Vorlesungen," Wien, 188.5, Bd. i. S. 312; Cohnheim, Firchow's Archiv, 1863, Bd.
xxviii. S. 241 ; Kiihne, " Lehrbueh der physiol. Chem.," 1866, S. 39, 51, 52 ; Sheridan Lea,
Journ. Physiol., Cambridge and London, 1890, vol. xi. p. 226.


the osmotic pressure of the dissolved products. This water may, of
course, be removed by subsequent evaporation at a low temperature, to
avoid injuring the ferment, and again dialysing; but practically the
diffusive power of the usual products of digestion is so low as to render
a process of alternate dialysis and evaporation a tedious and almost im-
possible method of freeing the solution completely of the products of
digestion. This action of the accumulated products of digestion renders
all digestive experiments carried out in glass essentially different from
those which go on within the alimentary canal, where the products of
digestion are removed as fast as they are formed. Not only must the
natural process run more quickly, but there is no reason for assuming
that it will even run qualitatively along the same lines. To take as an
example the tryptic digestion of proteids. There are formed, as we shall
see later, as end products, certain amido-acids, and a substance known as
antipeptone, but long before these products are finally reached, soluble
bodies are formed which can be shown to be capable of absorption and
assimilation by the epithelial cells lining the intestine.

Digestion experiments in vitro teach us the effects of digestion alone,
sundered from its constant companion in the natural process — absorption ;
and no perfect method has hitherto been devised whereby the effects of
these two processes working in conjunction can be demonstrated. In
the animal body the pure effect of digestion and absorption cannot be
observed by studying the chemical composition of the intestinal contents
and that of the contents of the channels of absorption, because the pro-
ducts of digestion are not merely absorbed by the lining cells, but are
profoundly modified by them in the process. Nor can the combined
effect of digestion and absorption be studied in perfection by any known
method of digestion and dialysis, because no artificial dialyser bears any
but a very remote resemblance to the living intestine. A dialyser of
parchment paper not only removes diffusible substances with infinite
slowness compared with the intestinal epithelium,^ but it also acts on
purely physical laws, diffusion taking place at rates directly proportional
to the diffusion coefficients of the substances involved ; while the living
epithelium takes up with great avidity soluble substances which do not
diffuse at all, and absolutely refuses passage to other very diffusible sub-
stances, such as soluble salts of iron. That is to say, absorption by the
cell is selective,, being governed, indeed, by fixed and definite laws, pro-
bably purely physical and chemical at bottom, but j)rofoundly modified
by the action of living protoplasm.^

The effects of removal of products of digestion by dialysis has been studied
by Sheridan Lea,-^ in tlie case of starch digestion by ptyalin, and proteid
digestion by trypsin. The rapidity of dialysis was increased by mechanically
raising and lowering the dialysing tube, and the rate of digestion and nature
of products formed were compared with those in an exactly similar experiment
arranged in a glass vessel. It was found (1) that the speed of digestion was
in all cases increased, and (2) that before the process came to a standstill
much more conversion took place than it was possible to attain to in glass,
although complete conversion never took place in either case ; these differences
were in every case more marked when concentrated solutions of the material
to be digested were used, showing that the slower digestion and earlier stoppage

^ Heidenhain, Arch. f. d. ges. Physiol., Bonn, 1888, Suppl. Heft, Bd. xliii. S. 60.
- For a furthei' consideration of this subject, see " Proteid Absorption," p. 430.
^ Journ. Physiol., Cambridge and London, 1890, vol. xi. p. 226.

VOL. I. — 21


in glass was due to accumulation in the solution of digested products. Similar
experiments on the digestion of various forms of proteid, by pepsin and hydro-
chloric acid, dialysing into hydrochloric acid of equal concentration, have been
made by Chittenden and Amerman,^ who found that removal of the products
of digestion did not essenfAally favour peptonisation or alter the relative
amount of albumose and peptone formed.

Concentration of enzyme. — The rapidity with which zymolysis takes
place naturally varies with the concentration of the enzyme in the
solution, as well as with the concentration of the material to be digested,
ivlien this is soluhle. Roberts found in the case of conversion of starch
by the diastatic enzyme of the pancreas, that the amount of standard
starch mucilage which can be converted in a given time and at a given
temperature varies directly as the quantity of active solution employed.

Schiitz ^ found in the digestion of proteid by pepsin, that when the
solutions employed were sufficiently dilute, the amount of conversion
was proportional to the square roots of the quantities of pepsin present.
Any such rule can only hold within certain limits of concentration, a
maximum being reached beyond which further concentration of the
enzyme has no effect.

Methods of estimating the relative activity of digestive solu-
tions. — As none of the enzymes have been isolated in a pure condition,
it follows that there is no means of estimating the absolute amount of an
enzyme in solution. This is practically never a matter of any moment,
but a problem which often presents itself in practical w^ork on digestion
is that of estimating the relative activities of two digestive extracts.

The activity of a diastatic enzyme can be most accurately estimated
by determining the amount of sugar (maltose) formed under given con-
ditions in a given time by a given volume of the solution, acting on a
measured volume of a standard solution of starch mucilage ; this, however,
is a tedious and troublesome process, and for most purposes a sufficiently
accurate process is that of observing when the starch has all disappeared,
as shown by the failure of the iodine reaction.

Such a method has been introduced by Roberts.^ He varies the amount
of the diastatic solution added until the " achromic poiyit " is reached within a
period lying between the limits of four and six minutes. This achromic point
is that point at which the starch solution ceases to give a yellow tinge with
iodine, when accordingly the solution contains only achroodextrins and maltose.
Roberts defines the diastatic value of a solution (denoted by the symbol D)
by the volume in cubic centimetres of a standard starch mucilage which can
be converted to the achromic point by 1 c.c. of that solution, acting during five
minutes at a temperature of 40° C.

The standard solution of starch mucilage must he prepared fresh ; it is
made by stirring up 5 grms. of pure potato starch with 30 c.c. of water, and
pouring slowly into nearly 470 c.c. of water, which is kept boiling. The
mixture is stirred and boiled for a few seconds, and finally accurately made up
to 500 c.c, thus giving a standard solution (1 per cent.) of starch.

The solution of iodine used is made by diluting 1 part of the liq. iodi of
the Pharm. Brit, with 200 parts of water.

In making a determination, one proceeds as follows : — Ten c.c. of the
standard starch mucilage are diluted with distilled water to 100 c.c. and

1 Journ. Physiol., Cambridge and Loudon, 1893, vol. xiv. p. 483.
' Ztschr. f. xihijsiol. Clicm., Strassburg, 1885, Bd. ix. S. 577.
^ Diastasimetiy, In " Digestion and Diet, " Loudon, 1891, p. 68.


warmed to 40° C. ; a known volume of the diastatic solution to be tested is

next added, say 1 c.c, noting the time; drops of the solution are then tested

from time to time, say at intervals of ten seconds, with drops of iodine on a

porcelain slab until no yellow tinge is produced, and the interval of time

which has elapsed is noted. By altering the amount of diastatic solution

added, as a result of preliminary experiment, this time must be arranged to lie

between four and six minutes ; if the time is shorter than four minutes, an

error of a few seconds in determining the time of conversion makes too large a

percentage error, or if it be much longer than six minutes the transition is too

gradual at the end for the eye to accurately catch the acliromic point. If v

be the volume in cubic centimetres of diastatic solution added, n the time to

reach the achromic point in minutes, and D the diastatic value of the solution

10 5
as above defined — then, D= — x —

V n

This value of D gives a measure of the activity of a given diastatic solution,

in terms of a standard which can be easily reproduced at any time to measure

the activity of another diastatic solution, and so comparable results may be


Various methods are in use for determining the relative activity of
proteolytic solutions.

The earliest method is that first introduced by Bidder and Schmidt, and
used in various modifications by other experimenters. It consists in deter-
mining the weight of proteid dissolved in equal times, by equal volumes of the
digestive liquids added to equal volumes of a proper digestive medium. The
method is oftenest used for relative determinations of pepsin, when the
medium used is hydrochloric acid solution of 1 or 2 per mille, but it may also
be used for trypsin, when \ per cent, sodium carbonate can be used as a
medium. The digestive solutions are placed in "% bath at 40° C, and when
they have acquired the temperature of the bath, equal weighed portions of
equally finely subdivided hard-boiled white of egg (obtained by passing through
gauze netting) are added to each, and digestion allowed to proceed for the same
period in each case, say twenty-four hours ; the liquids are then filtered, and the
residues left undigested are washed, dried, and weighed ; a third equal quantity
of the white of egg used is also dried and weighed without previous digestion ;
and from the figures so obtained the amounts of dissolved white of egg are
deduced, and these are taken as representing the comparative peptonising
values of the two samples.

Brilckes ^ method. — This method consists essentially in diluting the
two proteolytic solutions to be compared with the same medium (1 per
mille HCl) in two series, and then picking out those two members in
each series which are most nearly equal ; from the relative dilution of
these tw^o the comparative activity of the two original solutions easily

Vessels. Pepsm Solution of Acidity,

1 per Mille.





iVater of Acidity,

1 per Mille.







1 " Vorlesungen ueber Pliysiologie, " AVien, 1885, Aufl. 4, Bd. i. S. 311.


Hydrochloric acid is added to the two pepsin sokitions, iiiitil the acidity
represents 1 grm. of hydrochloric acid per litre. These are then diluted in a
series of vessels with hydrochloric acid (1 per mille) according to the foregoing
scheme ; the figures represent volumes, say cuhic centimetres.

A corresponding series of dilutions of the second solution is also prepared,
and in the vessels of both series a shred of fibrin ^ is digested for a given time.
At the end of the time, correspondingly advanced specimens are picked out in
the two series, especial attention being paid to the more dilute samples, which
give the truer indications, and the comparative power of the two solutions
easily follows. For example, if JN'o. 3 in one series corresponds to ]S[o. 5 in the
other, the latter is four times as powerful as the former ; a closer approximation
can then evidently be obtained by a second experiment.

Grunliageii's - method. — Fibrin is swollen out by placing it for some hours
in dilute hydrochloric acid. Equal weighed portions of this swollen fibrin are
IDlaced in similar filters. Over each portion an equal volume, say 1 c.c, of the
various digestive solutions to be compared are poured. Soon the fibrin begins
to dissolve and drop from the funnels, dissolving in the dilute acid which had
previously swollen it. From the measured amounts dropping in equal times
from the different funnels, or by counting the rate of the drops, the compar-
ative activities of the various solutions can be determined. This method
evidently cannot be used for trypsin.

Griltzner's ^ method. — Also cannot be used for trypsin, but is one of the best
methods for pepsin. It is a colorimetric method, and consists in measuring
the velocity with which the solution under examination dissolves fibrin stained
uniformly with carmine, by means of the depth of tint imparted to the solution
by the finely divided particles of carmine, which are set free in the solution at
a rate proportional to that of solution of the fibrin.

The method is best carried out by comparing the depth of the tints
produced at observed time intervals Avith those of a number of standard solu-
tions of carmine. The methods employed in preparing the stained fibrin and
these standard tints are as follows : — The fibrin is first well washed in a stream
of running water accompanied by kneading,'^ and then placed for twenty-four
hours in a bath of weakly ammoniacal 0"25 per cent, carmine solution,^ the
volume of staining fluid being large compared with that of the mass of fibrin
to be stained, and the latter being pulled into small pieces, so as to ensure
thorough and uniform staining. After staining for twenty -four hours, the
fibrin is removed from the staining bath and washed well in a stream of
running water until it ceases to colour it. Before using for a digestion experi-
ment, the coloured fibrin in small pieces is immersed in about five times its
volume of 0"2 per cent, hydrochloric acid for thirty to sixty minutes; this
swells it up to a clot-like mass, and it is used in this condition, pieces of
approximately equal size being placed in equal volumes of the various digest-
ive fluids to be compared, contained in equal-sized test tubes.

The scale of comparison tints may be prepared by adding, in varying pro-
portion, a glycerin solution containing one-tenth per cent, of carmine, to water
in test tubes of equal size; thus, to 19'9 c.c. of Avater are added 0"1 c.c. of
one-tenth per cent, glycerin-carmine solution; to 19 "8 c.c. of water, 0'2 c.c. of
the same glycerin-carmine solution ; and so on, finishing with a solution

^ Approximately of equal size ; a slight difference has no apprecdable effect.

^ Arch. f. d. ges. Physiol., Bonn, 1872, Bd. v. S. 203. For a method of adopting this to
experiment at body temperature, see Grlitzner and Ebstein, ibid., 1874, Bd. viii. S. 122.

^ Ibid., 1874, Bd. viii. S. 452 ; "Neue Uutersuch. li. Bildungu. Ausscheid. des Pepsins,"
Habilitationsschrift, Breslau, 1875.

* It may advantageously be left in water over night to I'emove accompanying ha>mo-

^ Prepared by dissolving 1 grm. of carmine in a small volume of dilute ammonia and
making up to 400 c.c. with water ; the solution should only very faintly smell of ammonia,
and if necessary must be left exposed to the air until the odour of ammonia almost disappears.


of 19 c.c. of water and 1 c.c. of glycerin-carmine solution. In this manner
ten standard tints are obtained, the values of which correspond to the numbers
1 to 10; these are mounted in a stand against a uniform white background,
and are used to compare with the results of digestion, after equal intervals
of time. For example, if after thirty minutes' digestion the tint of one test
tube corresponds most closely to that of Standard 2, while that of another
corresponds to Standard 6, the latter is three times as powerful a digestive
solution as the former. The digestive solutions should be so diluted that they
act somewhat slowly, because after a time a maximum tint obtains, and then
the weaker digestive fluid catches up on the other ; the farther apart from
this maximum the measurements are taken the better. Also, if a close approxi-
mation to the comparative amounts of pepsin in two solutions is required,
after a preliminary experiment the stronger of the two must be diluted
experimentally until its action is equal to that of the other, then the pro-
portion of dilution gives the proportionate strength in pepsin of the two

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 45 of 147)