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

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gives for fibrin 0-8 to 1 ; for myosin, casein, and vegetable proteid, 1 ; for liard-boiled proteid,
2-5 parts per litre. — "Lehrbucli," Aufl. 3, S. 238.


addition of hydrochloric acid, because the compound is not re-formed,
meets a simple answer in the statement that the pepsin is permanently
destroyed by the strong acid or alkali used. A strong argument against
any such compound is that the concentrations of different acids causing
equal activity in pepsin are not proportional to the chemical equivalents
of the acids, as might be expected if the acids entered into chemical
combination with the pepsin.^

Pepsin is very rapidly destroyed by solutions of alkalies or alkaline
salts.2 The principal conditions which influence the rate of destruction
of pepsin by sodium carbonate are — the strength of the solution of the
alkaline salt, the time during which it is allowed to act, the temperature
of the mixture, and the amount of proteid present. The mere act of
neutralising an acid pepsin solution may destroy a considerable part of
the pepsin. When equal volumes of a fluid containing pepsin and of a
1 per cent, solution of sodium carbonate are well mixed, the greater part
of the pepsin is destroyed in fifteen seconds ; in a neutrahsed acid
extract of the gastric mucous membrane of a cat, the amount thus
destroyed may be |^ of the whole. Even very dilute sodium carbonate
("005 per cent.) will cause an appreciable destruction of pepsin in one
or two hours at the body temperature, provided proteids are present
in small amount only.

Proteids lessen the rate of destruction of pepsin, probably by
combining with the alkah or alkahne salts, for the greater the amount
of sodium carbonate present the greater must be the amount of proteid
to lessen appreciably the destruction. In the presence of '5 per cent,
sodium carbonate, less than "25 per cent, of peptone has very little effect,
and even 2'5 per cent, of peptone does not prevent the greater part of the
pepsin from being destroyed. Thus, in the presence of 2 '5 per cent,
peptone, seven-eighths of the pepsin in an extract of a cat's gastric
mucous membrane may be destroyed at 17° C. by "5 per cent, sodium
carbonate in sixty seconds. Pepsin prepared from a frog is less rapidly
destroyed than pepsin prepared from a mammal. Carbonic acid destroys
pepsin also, but less rapidly than it destroys pepsinogen.^

Solutions of salts of the heavy metals weaken or entirely remove the
activity of pepsin solutions, according to the amount added. This effect
is probably due to the enzyme being mechanically carried down in the
usual fashion by the precipitate formed betw^een heavy metal and pro-
teid. The neutral salts of the alkalies and alkaline earths, when added
(even in small quantity) to solutions of pepsin^ decrease the activity.
Thus Al. Schmidt * found that salt-free proteid dissolved in a few seconds
in salt-free pepsin solution, but on the addition of 0'5-0'6 per cent, of
sodium chloride the time of solution was increased three to ten times.
Hydrobromic and hydriodic acids in large doses, and to a still greater
extent their potassium salts, delay peptic digestion. Sulphurous acid stops
peptic digestion, but arsenious and hydrocyanic acids, except in large
amounts, have little effect. Carbolic acid in small quantities has also
little effect, but acts injuriously in greater concentration. Salicylic acid

^Davidson n. Dietrich, Arch. f. Anat. «. Physiol., Leipzig, 1860, S. 688; Putzeys,
Jahrcsb. ii. d. Fortschr. d. Thier-Chem., Wiesbaden, 1877, Bd. vii. S. 279; Halm, Firchoio's
Arehiv, 1894, Bd. cxx.wii. S. 597.

^ Langley, ./ourn. Physiol., Cambridge and London, 1882, vol. iii. )ip. 253, 283 ;
Langley and Edkins, ihid., 1886, vol. vii. p. 371.

■' (.^Uioted from Langley and Edkins, loc. cit.

■^ Jahresh. il. d. Fortschr. d. Thier-Oheon., \A^iesbadcn, 1876, Bd. vi. S. 23,


was formerly credited with a powerful checking action, hut, as shown
by Klihne/ while powerful in preventing the growth of Ijacteria, this
acid has no appreciable action, especially in small quantity, on the un-
organised ferments. Pepsin is much more rapidly destroyed by standing
under strong alcohol than are the other enzymes.

Anything which prevents swelling of the proteid Ijy the acid retards
the progress of peptic digestion. Brticke ^ states that fibrin tied tightly
round with a thread, so that it cannot be so easily swollen by the acid,
is digested much more slowly. Adding a sufficient amount of neutral
salt also slows the digestion, probably from a similar cause, the salt
preventing the imbibition of acid by the fibrin. The comparative slow-
ness of digestion of heat-coagulated proteid, such as coagulated white of
egg, may also be due to a like cause, for such a form of proteid does not
swell up with acid. Finally, stronger acid than the optimum strength
does not cause so much swelling, and this may in part be the reason of
the slowing due to this cause.

Variation in rapidity loith form of proteid. — The time of digestion
by pepsin varies enormously with the nature and condition of the pro-
teid to be digested ; coagulated white of egg requires almost as many
hours as unboiled fibrin does minutes. The comparative rate of pepton-
isation of coagulated and non-coagulated white of egg has been much
investigated, and with varying results. According to AYaurinski,^ these
variations are due to want of uniformity in the concentration of acid
employed as a digesting medium ; with more dilute acid the coagulated
proteid is much more quickly digested, but the reverse is true when acid
of greater concentration is used.

The comparative speed of peptic digestion of different kinds of pro-
teid has, because of its practical bearing, been made the subject of much

Casein is the most easily digested of all forms of proteid. Fibrin is
much more quickly digested than coagulated egg-white, though, according
to its state of aggregation and time of boiling, the latter shows a con-
siderable variation. In general, proteids of animal origin are more
easily digested than those of vegetables, and of the latter legumin is
most easily, glutin most difficultly, digestible.^ Jessen ^ observed that
muscle fibre is more rapidly dissolved when raw than when coagulated
by boiling or roasting, and that boiled milk is digested more slowly than
unboiled. Beef appears to be both more easily dissolved and peptonised
than fish.''

The conclusion ought not, however, to be too hastily drawn that
those forms of proteid which are most easily dissolved by gastric juice
are therefore best and most nutritious ; gastric juice is not the only
proteolytic fluid which acts on the food. If the food has been properly

1 Verliancll. d. naturh.-oncd. Ver. zu Heidelberg, 1876, N. F., Bd. i. S 90.

2 "Vorlesungen," Wien, 1887, Aufl. 4, Th. 1, S. 312.

^ Jahrcsb. u. d. Fortschr. d. Thier-Chem., Wiesbaden, 1873, Bd. iii. S. 175.

"^ Besides those quoted below, see Stntzer, Ztschr. f. iiliysiol. Chem., Strassbiirg 1885
Bd. ix. S. 212; 1886, Bd. x. S. 153; 1887, Bd. xi. S. 207; 1888, Bd. xii. S. 72; Pfeiffer,
ibid., 1887, Bd. xi. S. 1; WolflF, Landwirthsch. Jalirb., 1890, Bd. xix. S. 795; Hahn'
Virchow's Arehiv, 1894, Bd. cxxxvii. S. 597.

^ Maly, in Hermann's " Handbuch," Bd. v. (2) S. 79.

^ Ztschr. f. Biol., Miincben, 1883, Bd. xix. S. 129. See also Bergeat, iMd., 1888, Bd.
xxiv. S. 139.

■^Chittenden and Cummins, Am. Chem. Journ., Baltimore, 1884, vol. vi. ISTo. 5;
Popoff, Ztschr. f. physiol. Chem., Strassburg, 1890, Bd. xxiv. S. 524.


masticated, it is not necessary that it should be dissolved before leaving
the stomach. It does not follow that the foods which are more rapidly
dissolved are also more rapidly peptonised, nor, indeed, that those which
are more rapidly peptonised are also more thoroughly utilised by the

Rennin.- — The presence of a milk-curdling principle in the stomach
of the calf has been known for ages, but it is only within recent times
that it has been shown that this action is due to the presence of a
soluble ferment or enzyme.

This enzyme is present in neutral aqueous infusions of the mucous
membrane of the stomach of the calf and sheep, but in the case of other
mammaha, of birds and of fishes, the zymogen is more stable, and the
active enzyme itself is only set free on treating a neutral infusion with

The presence of rennin in the stomachs of birds and fishes is very
remarkable, and points to some wider function of the enzyme, at present
unknown to us, since it cannot be supposed that in such animals the
ferment plays any part in connection with the clotting of milk. Many
plant juices also contain enzymes which coagulate milk, such as the latter
of the fig tree,* and of Carica iMiJciya, and the flowers of many cynaria.
Milk^ is also coagulated by bacterial action with the development of
an acid reaction due to lactic acid (in the souring of milk). A curdy
precipitate somewhat resembhng a clot is caused by the addition of acids
to milk, which led to the erroneous analogy being drawn, that the
coagulation of milk by rennet was also an acid action, due to lactic acid
set free from the lactose of the milk by ferment action.

The following is a summary of the proofs that milk coagulation is not an
acid action, but due to a specific enzyme (rennin), which acts on a proteid
(caseinogen) of the milk : — 1. When a neutral solution of rennin (rennet) is
added to alkaline milk, and the mixture is kept at 38°-40° C, complete
coagulation occurs in 4-10 minutes, and in the process the reaction remains
unclianged. 2. Solutions of caseinogen prepared from milk and free from
lactose coagulate in presence of calcium salts, on the addition of rennin.
3. Purified solutions of rennin have no action whatever on lactose.*'

Eennin is always present under normal conditions in human gastric
juice, both at birth and in the adult.''' The distribution of the enzyme
and its zymogen in the gastric mucous membrane is similar to that of

^ See "Absorption of Proteids," p. 441.

^ The name is due to Sheridan Lea ; that of chymosin has been proposed by Deschamp.

^ Hammarsten, "Lehrbuch d. physiol. Chem.," Wiesbaden, 1895, Aufl. 3, S. 241.

* This also contains a proteolytic ferment, active in either alkaline, neutral, or acid
reaction (Baginsky, Ztsclir. f. jjhysiol. Chem., Strassburg, 1882, Bd. vii. S. 209 ; Arch. f.
Anat. u. Phy-'iioL, Leipzig, 1883, S. 276).

^ For the chemistry of milk, see p. 125.

® These proofs are due to : Heintz, Journ. f. praTct. Chem., Leipzig, 1872, N. F., Bd. vi.
S. 374 ; Hammarsten, Jahresb. ii. d. Fortschr. d. Thier-Chem., Wiesbaden, 1872, Bd. ii.
S. 118 ; 1874, Bd. iv. S. 135 ; 1887, Bd. vii. S. 158 ; "Zur Kenntniss des Caseins und der
Wirkungdes Labfermentes," Upsala, 1877 ; Al. Schmidt, Jahresb. ii. d. Fortschr. d. Thier-
Chem., Wiesbaden, 1874, Bd. iv. S. 154. From the fact that rennet when impure acts on
lactose, but not after purification, Hammarsten sujiposed that gastric juice contained a
third enzyme, which acted on lactose, forming lactic acid, but this has not been sub-

' Zweifel, Cenlralhl. f. d. med. Wissensch. , Berlin, 1874, Bd. xii. S. 939 ; Hammarsten,
Beitr. z. Anat. u. Physiol, als FesUjahe C. Ludioig, Leipzig, 1874 ; Schumberg, Virchoiv's
Archiv, 1884, Bd. xcvii. S. 260; Boas, Centralhl. f. d. ined. Wissensch., Berlin, 1887, Bd.
XXV. S. 417.


pepsin ; that is, the pyloric part furnishes very weak extracts compared,
with those yielded by the fundus.^

Solutions of rennin, commonly called rennets, may be prepared Ijy
extracting the mucous membrane of the stomach in various ways, of
which the following is a summary : —

1. Extraction of the mucous membrane of the stomach of the calf for some
days with glycerin. Purer solutions may be afterwards obtained by precipi-
tating the glycerin extract with excess of alcohol, tiltering, and treating the
precipitate with Avater.

2. Digesting the mucous membrane of the stomach for twenty-four hours
at atmospheric temperature Avith water containing 1 to 2 parts per mille of
hydrochloric acid, filtering, and neutralising.

3. Extracting with a saturated aqueous solution of salicylic acid, precipi-
tating by excess of alcohol, and extracting the precipitate with water.

4. The best extractive for making permanent preparations is solution of
sodium chloride of from 5 to 15 per cent, concentration, putrefaction being
prevented by the addition of alcohol, thymol, or some such innocuous pre-

Effects of temperature. — Eennin is quickly destroyed in neutral solu-
tion by a temperature of 70° C, in acid solution by a temjjerature of
63° C. The temperature of maximum activity lies at 38 to 40° C. It
also acts, though more slowly, at atmospheric temperatures.

Action of acids and alkalies. — Eennin is rapidly destroyed by caustic
alkalies; even 0'025 per cent, of caustic soda suffices, at atmospheric
temperature in twenty-four hours, to completely destroy a very active
solution. The amount of ferment so destroyed varies as usual with the
duration of the action, the temperature, and the concentration of the
destructive agent. In their behaviour towards alkaline carbonates
rennin and its zymogen closely resemble pepsin and pepsinogen ; rennin
being quickly destroyed by 0-5 to 1-0 per cent, of sodium carbonate
(NaoCOg), while its zymogen is much less readily affected thereby.^

Eennin is destroyed by standing under alcohol, but this change occurs
more slowly than the corresponding one in the case of pepsin.

Separation of pepsin and rennin.— For the preparation of a pepsin
solution free from rennin, a gastric extract containing both enzymes is
submitted to digestion in 0-3 per cent, hydrochloric acid for forty-eight
hours at 38° to 40° C. ; the rennin is completely destroyed. Hammarsten ^
utilises Briicke's principle of mechanical precipitation, for the preparation
of a rennin solution free from pepsin, in the following method. An acid
infusion of the gastric mucous membrane is neutralised with magnesium
carbonate, and enough neutral acetate of lead is added to completely
precipitate all the pepsin accompanied by a portion of the rennin.* The
filtrate is further precipitated by more lead acetate aided by ammonia,
and the precipitate is separated and decomposed by very dilute sulphuric
acid, so }delding a solution of rennin almost free from proteid. This
solution is then further purified by mechanical precipitation with

The final product so obtained produced no effect on a flock of fibrin

^ Hammarsten, loc. cit.

2 Langley, Journ. Physiol., Cambridge and London, 1880-2, vol. iii. p, 287; Boas, Ztschr.
f. hlin. Med., Berlin, 1888, Bd. xiv. S. 249.

^ Loc. cit. * As tested by the inability of the filtrate to digest fibrin.


in twenty-four hours, but caused coagulation of fresh milk of neutral
reaction in one to three minutes.

Such a solution of purified rennin behaves essentially differently in
its reactions from a proteid solution. It is not coagulated by heat,
does not give the xanthoproteic reaction, and is not precipitated by
alcohol, tannin, iodine, or neutral acetate of lead ; it is, however, pre-
cipitated by basic acetate of lead.

The Pancreatic Enzymes.

The pancreatic juice of all vertebrates in which it has been tested ^
contains three distinct enzymes, each of which acts on a different class
of the three great divisions of foodstuffs.^ In the invertebrates generally,
the place of the pancreas is taken by the so-called liver, hepato-pancreas,
or digestive gland. This usually contains enzymes, capable collectively
of attacking all three classes of foodstuffs, and with varying reaction ;
so that this organ may be considered as taking the place of the combined
dij^estive elands of the vertebrates.^

The different enzymes of the pancreas do not appear equally early ni
life ; the pancreatic diastase, amylo2Jsin, is not found at birth, but first
appears a month or more afterwards.^ The proteolytic ferment, trypsin,
is found during the last third of foetal life.^ No similar information is
on record regarding the fat-splitting ferment, steapsin.

The relative amounts of the different enzymes in pancreatic juice
vary considerably. In passing from a flesh to a bread-and-milk diet, the
proteolytic activity is said to diminish while the diastatic activity in-
creases, and vice versd in passing from a carbohydrate to a proteid

In addition to the methods of extraction already described under
general methods, the pancreatic enzymes may be obtained in solution by
various other methods, of which the following is a summary : —

1. By extracting with water saturated with chloroform ; such an extract
keeps well and is very efficient.'^

2. By extracting with water containing 3 to 4 per cent, of a mixture of
2 parts of horacic acid and 1 part of borax. ^

3. By placing the fresh gland^ finely minced, in a saturated sohition of
sodium chloride. This gives a strong solution of the proteolytic and diastatic

4. By extracting the fresh pancreas, freed from fat and finely minced,
with about four times its weight of 25 per cent, alcohol for four or five days ;
succeeded by filtration, which may be assisted by a trace of acetic acid.^°

1 For a detailed account of tlie action of pancreatic extracts in different animals, see
Harris and Gow, Journ. Physiol., Cambridge and London, 1892, vol. xiii. p. 469.

- A milk curdling enzyme is also present ; see Milk, p. 127.

2 Krukenberg, " Grundzllge eiuer vergleichenden .Physiologic der Verdauuug," Heidel-
berg, 1882.

■* Korowin, Jaliresh. ik d. Fortschr. d. TJiier-Chcm., Wiesbaden, 1873, Bd. iii ; Zweifel,
"Untersuch. ueber den Yerdauungsapparat der Neugeborenen," Berlin, 1874.

•'' Albertoni, Jaliresh. u. d. Fortschr. d. Thier-Chem., Wiesbaden, 1878, Bd. viii. S. 254.

^ Vassiliew, Arch, de sc. liol., St. Petersbourg, 1893, vol. ii. p. 219; Jahresb. ii. d.
Fortschr. d. Thicr-Chem., Wiesbaden, 1893, Bd. xxiii. S. 219.

'' Roberts, " Lumleian Lectures," 1880 ; "Digestion and Diet," London, 1891, p. 18.

^ Roberts, loc. cit.

^ Roberts, loc. cit. Also recommended by Harris and Gow, Journ. Physiol., Cambridge
and London, 1892, vol. xiii. p. 469.

^" Roberts, loc. cit.


5. A very active proteolytic extract may be obtained by extracting Avith
water containing O'Ol to 0'05 per cent, of ammonia. The filtered extract gives
a precipitate with acetic acid which digests proteid very energetically, and can
be further purified. ^

In preparing pancreatic extracts, it should, be remembered that the
gland does not at all periods contain the same amount of the ferments,
or rather their zymogens, but that the amount fluctuates within wide
limits according to the period after a meal. The pancreas of an animal
in which digestion is not going on will yield little or no ferments ; the
best time is from four to seven hours after a meal. An inactive prepara-
tion may often be cured by making the extract faintly acid with acetic
acid some time before using ; this sets free ferment which may be present
as zymogen in the extract.

Trypsin.^ — The proteolytic enzyme of the pancreatic juice in the
purest form in which Klihne obtained it, gave all the proteid reactions,
thus differing from all the other purer forms of enzyme hitherto described.
Kilhne's product is decomposed on boiling, yielding 20 per cent, of
albumin and 80 per cent, of peptone ; it is soluble in water, but insoluble
in anhydrous alcohol or glycerin. The insolubility of the purified dry
product in anhydrous glycerin accords with v. Wittich's ^ observations,
that both enzymes can be extracted from the fresh gland by glycerin ;
but if the gland mass be previously thoroughly dried by extraction with
alcohol, glycerin only takes out the diastatic enzyme, the proteolytic
one being left behind.

Infiuence of temperature. — The activity of trypsin increases, accord-
ing to Eoberts,* with rising temperature until 60° C. is reached, and
then rapidly falls, all action ceasing between 75° C. and 80° C. ;
Biernacki^ states that purified trypsin in 0-25 to O'S per cent, sodium
carbonate solution is destroyed in five minutes by a temperature of
50° C, and in neutral solution by a temperature of 45° C. The presence
of albumoses or of certain ammonium salts protects against the action
of elevated temperature in alkaline solution.

Influence of reaction. — Kiihne ^ made the observation that the activity
of trypsin was permanently destroyed by digesting its solutions with
pepsin and hydrochloric acid, and attributed the greater share in this
action to the pepsin. Boas'^ afterwards showed that the destruction
might be due to acid action alone, by demonstrating that addition of
hydrochloric acid to the filtered intestinal contents causes a precipitate
containing nearly all the ferments. This precipitate, on standing for a
few hours under the acid, became inert, but, when quickly separated and
redissolved in sodium carbonate, showed both diastatic and proteolytic
action. The matter has recently been again tested by Melzer,^ who

1 Hammarsten, "Lehrbuch," Wiesbaden, 1895, Aufl. 3, S. 26.5.

^ So named by Klihne, Verhandl. d. nahirh.-med. Ver. zto Heidelberg, 1876, N. F.,
Bd. i. S. 190. Danilewski {Firchoiv's ArcMv, 1862, Bd. xxv. S. 279) had previously to this
obtained a product which failed to give many of the usual proteid i-eactions.

^ Arch. f. d. ges. Physiol., Bonn, 1869, Bd. ii. S. 198 ; Hlifner (Jahresb. u. d. Fortschr.
d. Thier-Chem., Wiesbaden, 1872, Bd. ii. S. 360) failed to obtain a similar result, probably
through using glycerin containing water.

"* Proc. Roy. Sac. London, 1881, vol. xxxii. p. 158.

s Ztschr.f. Biol., Mimchen, 1891, Bd. xxviii. S. 51.

^ Verhandl. d. naturh.-med. Ver. zu Heidelberg, 1876, N. F., Bd. i. S. 193.

^ Ztschr.f. klin. Med., Berlin, 1890, Bd. xvii. S. 170.

^ Inaug. Diss., Erlangen, 1894 ; Melzer's figures show that most of the destruction is due
to acid alone.

VOL. I. — 2 2


finds that hydrochloric acid alone does destroy trypsin, but not so
rapidly as when pepsin is also present.

All possible opinions have been held by various observers as to the
reaction with which trypsin acts, and acts best ; ^ it is now generally
accepted that it can act either in an alkaline, neutral, or very faintly
acid solution, but that the optimum reaction is that given by about 1
per cent, sodium carbonate (N'aaCog).^

Active proteolysis by trypsin cannot take place in presence of an
acid reaction, except the acid be combined with proteid. If the proteid
be completely saturated with acid, the rate is greatly slackened even
when there is no free acid in the solution ; and if much proteid be
present, the ferment action may be abolished even before this stage is

Heidenhain * states that the concentration of sodium carbonate
necessary to ensure maximum activity varies with the richness in
ferment of the solution' experimented upon ; the richer in ferment, the
lower the percentage of sodium carbonate necessary for maximum

Other alkaline carbonates are much less effective than sodium
carbonate in increasing the activity of trypsin. The action is also said
to be assisted, but to a still less degree, by other salts of the alkalies.^

Organic acids have not nearly so destructive an action as hydro-
chloric acids, arsenious acid has no hindering effect, and salicylic acid
only when in saturated solution.^

The nature of the proteid submitted to digestion by trypsin has
also a profound effect upon the rapidity of the process. Fresh unboiled
fibrin is so quickly dissolved that it cannot be used as a comparative
test for trypsin, and fibrin which has been boiled, or discs of hard-boiled
white of egg, must be substituted for it.

Amylopsin. — An active amylolytic extract of pancreas can best be
prepared by following Eoberts' method of extracting with dilute alcohol.

Pancreatic juice is much more intensely diastatic than saliva, but
it cannot be determined, until some method for isolating the diastases
has been discovered, whether this is due to a difference in the amylolytic
ferments present or to a mere difference in concentration. It is certain,
however, that salivary, pancreatic, and malt diastases are practically
identical in the qualitative character of their action on starch. Eoberts

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 47 of 147)