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

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solutions. This determination may be most speedily attained by making a
simultaneous series of dilutions of the stronger solution, and comparing the
strength of their action with that of the other solution or a series made
from it.

Two tubes of equal speed of action are picked out, and from their dilutions
the comparative richness in pepsin of the original fluid easily follows. Griltzner's
method may also be employed without a scale of standard tints, by stopping
digestion after an equal period, and then diluting the stronger solution until
its tint becomes equal to that of the weaker, or by carrying out two series
in aliquot dilution of the two solutions to be compared, and picking out
equally advanced members of the two series. In case the comparison is made
Avith solutions of unequal power, it must be remembered that what is measured
is the comparative digestive power and not the comparative strength of the
solutions in pepsin, because the two are not proportional ; ^ in all cases it is
preferable, for accuracy, to prepare solutions from the originals of equal power,
and from the amount of dilutions of these to deduce the comparative strength
in pepsin of the originals, as indicated above.

Mette's method.'^ — This method is stated by Samojloff to yield exact
results. It consists in filling fine glass tubes of 1 to 2 mm. in diameter with
fluid white of egg, then coagulating by heat, and cutting off pieces of equal
length. These are placed in the digestive solutions at body temperature, and,
after the lapse of a certain interval, the length of white of egg digested off is
measured, which gives a measure for the comparative activities of the two

G-rlitzner ^ has also introduced methods for comparing the diastatic
and fat-splitting powers of pancreatic extracts.

That for diastatic action closely resembles Griinhagen's method for proteo-
lytic action. Equal volumes of 3-4 per cent, starch paste are placed on similar
filters, through which they do not filter until dissolved ; to each filter 0-2 to
0"3 c.c. of the extracts to be compared are next added, when solution of the
starch takes place at a rate proportional to the amount of enzyme present,
and a comparison of the amounts filtering through in a given time supplies
a measure for the activities of the extracts.

The method of comparing the fat-splitting powers of different extracts
consists in allowing the extracts to act on an emulsion in presence of litmus,
and noticing the time and amount to which the latter is turned red by the
acid developed. The emulsion recommended is made by mixing 10 parts of

1 See Schiltz's law, p. 322.

^ Samojloff, Arch, da sc. hiol., St. Pi^tersbours;, 1893, tome ii. p. 707.

3 Arcli.f. d. ges. Physiol., Bonn, 1876, Bd. xii. S. 293, 303.



oil of almonds, 5 parts of gum-arabic, and 35 parts of water. A solution
of litmus is prepared of such concentration and reaction that it shows a violet
colour when placed in test tubes about a centimetre in diameter in front
of Avhite paper; in each test tube 10 c.c. of this dilute litmus solution are
placed ; to each five drops of emulsion are added ; then equal volumes of each
of the pancreatic extracts to be compared. From the times in which an equal
amount of red develops in the litmus in each case, the richness of the extracts
in fat- splitting ferment may be determined. Or a series of determinations
for each extract, using a varying quantity of it, may be made, and the
members of each series compared.

The condition of the material to be digested has also a profound
effect upon the rapidity. The factors of most moment are —

1. Whether the material is fluid or solid.

2. Whether it has previously been heated (cooked) or not. In the
case of starch, previous heating and formation of a starch paste shortens
the process in the ratio of hours to minutes ; in the case of proteids,
previous heat coagulation slows the after process of digestion.

3. Materials v^hich must first be dissolved, and must therefore be
attacked from the outside, are digested more quickly when in a finely
subdivided condition.

Classification of Enzymes.

Class of Enzj-me.

Name of Enzj-me.

Digestive Fluid in
which found.

Concise Description of
Specific Action.

Diastatic . . ■

1. Ptyalin

2. Aniylopsin

Pancreatic juice

Convert amyloses
(starches and glyco-
gen) into dextrins,
maltose, and isomal-
tose, accompanied by
a trace of glucose.

Proteolytic . . -

1. Pepsin

2. Trypsin

Gastric juice
Pancreatic juice

Converts proteids into
albumoses and pep-

Converts proteids into
albumoses, peptones,
and amido- acids.

Fat-splitting or

Steapsin or

Pancreatic juice

Splits up neutral fats
into fatty acids and

Coagulating . :

1. Renniu

2. An unnamed
ferment occur-
ring in ]3an-
creatic juice,
which also

Gastric juice

Coagulates milk, con-
verting caseinogen in
presence of calcium
salts into casein.


Specific action of enzymes.— The different enzymes are specific in
their action ; that is to say, each enzyme only acts on one class of
material and acts on it in a determinate manner, producing certain
specific snlistances as the result of that action. The table on p. 326 is
a classification of the digestive enzymes according to their specific action.

Description of the digestive enzymes.— -The digestive enzymes
may be here most conveniently treated of according to their occurrence
in the various digestive secretions, because of the description of the mode
of their separation, where more than one is found in the same digestive
fluid. Their action on the different classes of foodstuffs and the products
formed thereby will be considered afterwards.

Ptyalin.— In the saliva of many animals, and especially in the herbivora,
a diastatic enzyme is found, to which, soon after the discovery that saliva
possessed such an action,^ the name ptyalin was apphed. In fishes
and in cetacea no salivary glands are present,^ and in some other
animals the salivary secretion possesses no diastatic action ; for example,
the saliva of the dog has no diastatic action, and the same statement is
made for the typical carnivora in general.^ In man, the secretion of both
the parotid and submaxillary glands has a diastatic action. At birth the
ferment is only found in the parotid ; it makes its first appearance in
the submaxillary two months later.''^ In the horse the secretion leaves
the parotid with the diastatic ferment still in the condition of a
zymogen, from which the enzyme is set free by treatment with alcohol
or by contact with unfiltered air.^

Ptyalin was first separated from saliva in an impure form by
Mialhe,^ by precipitating filtered saliva with excess of absolute alcohol.
A scanty flocculent proteid precipitate is so obtained, which carries
down the ptyalin mechanically. Mialhe showed that this precipitate,
which was insoluble in strong alcohol, but partly soluble in water or
weak alcohol, possessed when dissolved the diastatic power of the
original saliva. From its supposed identity with the diastase of malt,
he called it diastase animal ou salivaire, and used the term ptyalin as a
synonym. It is now known that ptyalin and malt diastase, though
alike in their action upon starch, are not identical. This is shown best
by the difference in the reaction of the two enzymes to changes in
temperature. According to Eoberts,'^ saliva possesses a maximum
action between the temperatures of 30° and 45° C, and, according to
Kjeldahl,^ the optimum temperature is 46° C, while the enzyme is rapidly
destroyed by a temperature lying between 65° and 70° C.^ On the

1 Leuclis, Arch. f. d. ges. Naturl., Nlirnberg, 1831 ; Schwann, Ann. d. Pliys. u. Chein.,
Leipzig, 1836, Bd. xxxviii. S. 358.

^According to Krukenberg ("Grundzllge einer vergleicli. Phj^siol. der Verdauung,"
1882, S. 67), in some fishes the secretion of the mucous glands of the mouth possesses a
diastatic action; the same is true of the mucous secretion of the frog's mouth.

2 Grlitzner, Arch. f. d. ges. Physiol., Bonn, 1876, Bd. xii. S. 285 ; Bunge, "Lehrbuch
der physiol. Chem.," Leipzig, 1894, Aufl. 3, S. 140; Neumeister, "Lehrbuch der
physioh Chem., etc.," Jena, 1893, Th. 1, S. 122.

* Zweifel, " Untersuch. ueber den Verdauungsapparat. der Neugeborenen," Berlin,
1874. See also SchilTer, Jahrcsh. ii. d. Fortschr. d. Thier-Chem., Wiesbaden, 1872, Bd.
ii. S. 205 ; Korowin, ibid., 1873, Bd. iii. S. 158 ; Bayer, ibid., 1876, Bd. vi. S. 172.

° Goldschmidt, ZtscJir. f. 2^hysiol. Chem., Strassburg, 1886, Bd. x. S. 273.

** ComiJt. rend. Acad. d. sc, Paris, 1845, tome xx. pp. 654, 1483.

"" " Digestion and Diet," London, 1891, p. 79.

® Abstract in Jahresb. ii. d. Fortschr. d. Thier-Chem., Wiesbaden, 1879, Bd. ix. S. 381.

® Roberts, loc. cit. ; Kuhne states that saliva loses its activity at a temperature of
60° C. (" Physiol. Chem.," S. 21).


other hand, the optimum temperatm-e for malt diastase lies at 50° to
56° C. ; 1 the activity does not greatly diminish until 60° C. is passed, and
then rapidly decreases and disappears, the ferment being destroyed by a
temperature of 80° C. Malt diastase is also much more sensitive to the
presence of sahcyhc acid than is ptyalin, being stopped by the presence
of 0-05 per cent., while ptyalin is first affected by Ol per cent., and not
completely stopped until a strength of 1 per cent, is reached.^

There is unfortunately no such certainty as to the identity or non-
identity of ptyahn and amylopsin (the diastatic ferment of the pancreas),
which is also called ptyalin by some authors.^ By others, the two
enzymes are accounted different, because (a) the pancreatic action is more
intense and complete, and (&) there are certain differences in the
products formed by the action of the two enzymes.^ It is, however,
questionable whether these effects may not be entirely produced by
differences in concentration in the two cases of one and the same
ferment. In their behaviour to change of temperature and reaction the
two enzymes are identical ; the rate of conversion of starch into other
substances depends on the concentration of the enzymes in the solution ;
and with regard to differences in the products formed, it is not denied
that in prolonged salivary digestion a small quantity of dextrose is
formed, it is only claimed that larger quantities of dextrose are
formed in a shorter time ^ by the action of the diastatic enzyme of the
pancreas ; this again is a difference in degree and not in kind, and may
well be due to a difference in concentration of enzyme.

Cohnheim ^ obtained ptyahn in a purer form, that is, more free from
admixed proteids, by a method closely resembling that of Brlicke for
pepsin, and consisting essentially in producing a precipitate of tricalcic
phosphate in the saliva by the addition of phosphoric acid followed by
milk of lime ; this precipitates mechanically ptyalin and proteid, the
ptyalin dissolves more easily than the proteid on afterwards washing
the precipitate with distilled water, and may in this way be separated.

The solution so obtained was actively diastatic, but yd gave none of
the usual proteid reactions, was not coagulated on boiling, gave no
reactions with nitric acid, mercuric chloride, tannin, iodine, or acetic acid
and potassium ferrocyanide. The ptyalin precipitated from it was not
a pure substance, but contained chlorides and phosphates of sodium and

Excess of alcohol caused a flocky precipitate of phosphates, and an
amorphous granular substance coloured yellow by iodine. Dried at a low
temperature, this precipitate furnished a white powder, only slightly
soluble in water, which retained its diastatic action for months.

A very active material may also be obtained by v. Wittich's method

1 Chittenden and Martin, Stud. Lab. Physiol. Chem., jSTcw Haven, 1885, vol. i. p. 117 ;
abstract in .Jahresh. it. d. Fortschr. d. Thier-Chem., Wiesbaden, 1885, Bd. xv. S. 263;
Lintner and Eckhard, Journ. f. i^rakt. Chem., LeijDzig, 1891, IST. F., Bd. xli. S. 91 ; Stntzer
and Isbert, Ztschr. f. 'pliysiol. Chem., Strassburg, 1888, Bd. xii. S. 72.

2 Jul. Mtiller, Journ. f. prakt. Chem., Leipzig, 1875, N. F., Bd. x. S. 45.

^ Neumeister, " Lehrbuch der physiol. Chem.," Jena, 1893, Th. 1, S. 147.

■* See Sheridan Lea, "Chemical Basis of the Animal Body," London, 1892, p. 57.

^ Lea, however, found no dextrose, but oidy maltose, in his experiments quoted on p. 394.
See also Brown and Heron, Proc. Roy. Sac. London, 1880, No. 204, p. 393 ; Musculus and
Gruber, Ztschr. f. physiol. Chem., Strassburg, 1878-9, BA. ii. S. 177; Musculus and v.
Mering. iUd., S. 403 ; v. Mering, ibid., 1881, Bd. v. S. 185.

^ Virchow's Archiv, 1863, Bd. xxviii. S. 241. Compare Sundberg's statement as to
similar precipitation of pepsin l)y alcohol and not by tannic acid, p. 316.


of extracting the salivary glands with glycerin, precipitating the
glycerin extract with excess of alcohol, washing with strong alcohol,
and then extracting with water.

Effects of reaction. — A knowledge of the effects of change of
reaction on the amylolytic activity of ptyalin, apart from its intrinsic
interest, possesses considerable importance from the bearing it has on
the natural process of digestion of starch, and for this reason probably
the subject has attracted the attention of a great number of workers.^
Ptyalin is secreted in an alkaline fluid, the saliva, and after a few
seconds admixture with the food passes with it into the stomach ; here
its alkaline reaction is lessened by the gastric secretion, and finally
replaced by an acid reaction. The amount of starch changed by the
ptyalin will depend on the effect of this gradual diminution in alkalinity
on its activity, and if the activity is decreased thereby, on the rate at
which progress is made towards an acid reaction.

It was formerly supposed that ptyalin was only active in a fluid
of alkaline reaction, that it was in consequence only active during
the few seconds of mastication, while the food remained in the
mouth, and was instantly destroyed on coming in contact with
gastric juice. More recent observations have, however, shown that
the importance of saliva as a digestive fluid is much underrated by
such a view.

The diastatic action of ptyalin attains a maximum wdien the reaction
of the fluid containing it is neutral, or even faintly acid, provided the
acidity is due to acid combined with proteid. Even mere traces oifree
acid, however, lessen and rapidly destroy its activity. Sodium carbonate
added to neutralised saliva decreases its activity, and in greater
quantity arrests it ; here, again, proteids present in solution play a
protecting part, and by combining with the alkali prevent its injurious
action on the ferment. A solution of ptyalin free of proteid would
therefore probably act best in a neutral fluid, and would be quickly
destroyed by either an acid or alkaline reaction, due to acid or alkali
uncombined with proteid.^

The diastatic action of the saliva, therefore, continues in the stomach
during and after a meal until (1) the alkali of the saliva has been
neutralised, (2) the proteid present in solution has been satisfied, and
(3) a trace of free hydrochloric acid remains in excess. According

^ Jacubowitsch, Lehmann's " Zooohemie," in Gmelin's " Haudbnch der Cliem.,"
Heidelberg, 1858, Bd. viii. S. 22 ; Paschutin, Arch. f. Anat. u. Physiol., Leipzig, 1871,
S. 366 ; Hammarsteii, Jahresh. u. d. Fortschr. cl. Thier-Chem., Wiesbaden, 1871, Bd. i.
S. 35 ; Brlicke, Sitzungsh. d. k. ATcad. d. Wissensch., Wien, 1872, Abth. 3 ; "VVatson,
Trans. Chem. Soc, London, 1879, p. 539; Chittenden and Griswold, Am. Chem. Joxirn.,
Baltimore, 1881, vol. iii. p. 305 ; Falk, Jahresh. il. d. Fortschr. d. Thier-Chcm., Wiesbaden,
1881, Bd. xi. S. 444 ; Langle}'', Journ. Physiol., Cambridge and London, 1880-2, vol. iii.
p. 246 ; Nylen, Jahresh. il. d. Fortschr. d. Thier-Chem., Wiesbaden, 1882, Bd. xii. S.
241 ; Chittenden and Ely, Am. Chem. Journ., Baltimore, 1882, vol. iv. ; Journ. Physiol.,
Cambridge and London, 1882, vol. iii. p. 327 ; Detmar, Ztschr. f. physiol. Chem.,
Strassburg, 1882, Bd. vii. S. 1 ; Langley and Elves, Journ. Physiol., Cambridge and
London. 1883, vol. iv. p. 18 ; Chittenden and Smith, Chem. News, London, 1885, vol.
liii. ; Stud. Lah. Physiol. Chem., New Haven, 1885, vol. i. p. 1 ; John, Centralhl. f. Min.
Med., Bonn, Bd. xii.; Schlesinger, Virchoiv's Archiv, 1891, Bd. cxxv. S. 146 ; Schierbach,
Skandin. Arch. f. Physiol., Leipzig, 1892, Bd. iii. S. 344 ; Ebstein u. Schulze, Virchoiv's
Archiv, 1893, Bd. cxxxiv. S. 475. "

^ It is generally held that ptyalin acts best in neutral solution or with a faint acid
reaction, due to acid combined with proteid ; but there are slight dift'erences of opinion as to
where the exact optimum point lies, for which the original papers should be consulted,
especially those by Langley and by, Chittenden and their co-workers.


to the observations of van d. Velden ^ there is no free hydrochloric acid
found in the stomach until, on an average, three-quarters of an hour
after a carbohydrate meal. During this time the diastatic action of the
saliva must continue, and probably during most of the interval more
intensely than it would with its natural reaction. In this stage gastric
juice removed by the pump possesses a diastatic action on starch, but
later, when free acid is present, even when saliva is added to it, has no
such power. After all the proteid present in solution in the stomach
has been combined with the acid first secreted in the gastric juice,
and still more acid is secreted which remains free, the ptyalin not only
becomes inert, but is rapidly destroyed, and does not come into action
again after the acid of the gastric juice is neutralised in the small

Free, organic acids also act destructively on ptyalin ; the concentration
of acid required is greater than in the case of hydrochloric acid, and
varies with the particular acid as well as with the concentration of the
ferment in the solution. Different neutral metallic salts possess different
actions ; some diminish the activity, such as mercuric chloride, which
even in a concentration of 0'005 per cent, is sufficient to stop all action ;
others increase it when present in small quantity, such as magnesium
sulphate up to 0'025 per cent., but have an opposite effect in greater
concentration.^ Carbolic acid does not produce much effect, digestion
with 5 per cent, solution for some hours being required to destroy the

Pepsin. — Pepsin is very widely distributed in the animal kingdom ;
it is found in the gastric juice of all vertebrates, with the possible excep-
tion of some fishes.^ In the frog it is found chiefly in the oesophagus.*^
In the crayfish a yellowish-brown fluid is found in the mouth, of strong
acid reaction, which digests fibrin readily.'^ And in many insects an acid
proteolytic secretion has been observed. Similar acid proteolytic secre-
tions are also known in the vegetable kingdom, such as those which may
be obtained by stimulating the leaves of insectivorous plants.^ Whether
these acid proteolytic ferments of the invertebrates and plants are
identical with pepsin is not known with certainty, but they are very
similar in their action.

Pepsin is found in the stomach of the herbivora at birth, and in
some other animals, including man ; in others, it first appears two or
three weeks after birth, as in the dog and cat.^

The different regions of the stomach do not, on extraction, yield

]Ztsc]ir. f. 2}hysiol. Chem., Strassbiirg, 1879, Bd. iii. S. 205.

^ See Laiigley, Journ. Physiol., Cambridge and London, 1882, vol. iii. p. 246 ; Nylen,
Jahresb. il. d. Fortschr. d. Tlder-Chem., AViesbaden, 1882, Bd. xii. S. 241 ; and other
authorities quoted above. Opposite results were obtained by Cohnheim, Vircliow's ArcMv,
1863, Bd. xxviii. S. 248 ; Schiff, " Lec^ons sur la digestion," tome i. p. 162 ; and Diifresne,
Comft. rend. Acad. d. sc, Paris, 1879, tome Ixxxix. p. 1070.

^ Nasse, Arch. f. d. ges. Physiol., Bonn, 1875, Bd. xi. S. 138 ; Chittenden and Painter,
Shul. Lah. Physiol. Chem., New Haven, 1885, vol. i. p. 52.

^ Plugge, Arch./, d. ges. Physiol., Bonn, 1872, Bd. v. S. 550.

■'' Hammarsten, "Lehrbuch der Phy,sio]. Chem.," Wiesbaden, 1895, Aufl. 3, S. 234.

« Swiecieki, Jahresb. il. d. Fortschr. d. Thicr-Chem., Wiesbaden, 1876, Bd. vi. S. 172.

■^ Hoppe-Seyler, ibid., S. 170.

^ Darwin. "Insectivorous Plants" ; Goebeb and Loew, Chem. Centr.-Bl., Leipzig, 1893,
Bd. ii. S. 1065.

^ iMoriggia, Untersuch. z. Natnrl. d. Mensch. u. d. Thiere, 1876, Bd. xi. S. 455 ;
Hammarsten, Beitr. z. Anat. u. Physiol, als Fcstgabe C. Ludxcig, Leipzig, 1874, S. IIG ;
Zweifel, "Ueber d. Verdauiingsapparat der Neugeborenen," Berlin, 1874.


equal amounts of pe^jsin: the pyloric end always contains much less
than the fundus or the cardiac end, but is never quite devoid of pepsin.
It was formerly held by some observers that the pepsin found in the
pyloric end was due to infiltration by the secretion from the glands of
the remainder of the stomach, ]jut the secretion obtained from pyloric
fistula3 contains pepsin which can only be secreted by the glands of this
region of the stomach.^

Effects of temperature. — Pepsin in neutral solution is destroyed by a
temperature of 55° C. ; in a solution containing two parts per thousand
of hydrochloric acid it is not destroyed at this temperature, but is
destroyed in five minutes at a temperature of 65° C. By the addition
of peptones or certain salts it is so protected that it is only destroyed
in an equal time by a temperature of 60° C.^ According to v. Wittich,^
the maximum rapidity of action is found between 35° and 50° C, and
the rapidity of destruction by elevated temperature (as in the case of
ptyalin) is dependent on the amount of dilution of the ferment, and the
duration of the high temperature. The more dilute the pepsin solution
the more quickly it is destroyed, and the lower the limit of temperature
necessary. Pepsin is still faintly active at 0° C*

Effects of reaction. — Pepsin is only active in acid solution ; the most
effective acid is hydrochloric acid, but other acids are also capable of
setting it in action in varying degree. The most energetic of the other
acids are nitric, lactic, and phosphoric, followed at some distance by
sulphuric, acetic, oxalic, and tartaric acids. The most effective acids
seem also to be those which most easily swell up fibrin. Acid sodium
phosphate does not confer activity on pepsin.^

The amount of acidity required for optimum activity varies greatly
with the form of proteid to be digested; thus Brucke*^ gives for
fresh fibrin -08 per cent., but for heat-coagulated fibrin -12 to -16 per

Su2J]J0sed compound of pepsin and hydrochloric acid. — The hypothesis
has been put forward, that the pepsin and hydrochloric acid in gastric
juice are united to form a loose compound " pepsin - hydrochloric

There is no clear evidence in favour of the existence of such a
compound. It is said to be precipitated from gastric juice by the
soluble salts of lead and mercury, and to be re-obtainal3le unaltered
from the precipitate by decomposing with sulphuretted hydrogen. But
it is certain that both the acid and pepsin would be thrown down by
such salts, and there is no reason to suppose that they are not thrown
down separately instead of as a compound, and recovered together again
on decomposing the mixed precipitate. A second argument, that the
supposed compound acid can be decomposed by strong acids or alkalies,
and that the pepsin so separated does not again become active on the

1 See Ebstein and Grlitzner, Jahresh. ii. d. Fortsclir. d. Thier-Chem., Wiesbaden, 1872,
Bd. ii. S. 210 ; 1873, Bd. iii. S. 169 ; 1874, Bd. iv. S. 236 ; KlcmensieAvicz, ibid., 1875,
Bd. V. S. 162; Heidenliaiii, ibid., 1878, Bd. viii. S. 245 ; Khig, ibid., 1894, Bd. xxiv.
S. 334 ; Akermann, Skandin. Arch. f. Physiol., Leipzig, 1895, Bd. v. S. 134.

'^ Biernacki, Ztschr. f. Biol., Miiiieheii, 1892, Bd. xxviii. S. 49.

3 Arch./, d. ges. Physiol., Bonn, 1869, Bd. ii. S. 193 ; 1870, Bd. iii. S. 339.

^ Flanni, Ztschr. f. Biol., Mtinchen, 1892, Bd. xxviii. S. 453.

^ Maly, Hermann's " Handbuch," Bd. v. (2) S. 73.

^ Sitzungsb. d. k. Akad. d. Wissensch., Wien, 1859, Bd. xxxvii. S. 131 ; Hammarsten

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