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Enterokinase activating



Deuterase activating



Temp, coefficient
(10**)

Pancreatic juice 2*10\

Extract of sheep's pancreas (2*07, 1*93) 2-00 {

„ „ pig's pancreas (2-07, 1-97, 1-93,

1-93, 1-86, 1-81) II
„ „ ox pancreas 1-96'

rPancreatic juice 1-09 'j

~ pancreas M8|

100
1-22;



J Extract of sheep's
1 ., „ pig's
^ f» ft ox



200



112



A large number of experiments were made in which the data were in-
sufficient to give an accurate temperature coefficient, so in order to compare



200




20 30

TIME IN MINUTL^

Pig. 11.

the temperature effects the method adopted was to read off from the plotted
curves the times taken for liberation of 50 % of the total amount of trypsin
set free in 30 or 40 minutes at 37°, and then find the time taken for the
liberation of the same amount of trypsin at 17°. The time of liberation
of 50 % of trypsin is taken for comparison rather than that of the completion
of activation, as it can be estimated more accurately.

The differences of temperature effect observed with extracts of pig's
pancreas and of sheep's pancreas appear to depend on the quality of both
enzyme and substrate. Taking first the experiments made with glycerin
extracts, it was found that when pig's pancreas deuterase activated sheep's
pancreas trypsinogen, it took 1-4 times longer at 17° than at 37°, instead
of 1*5 to 3*1 times longer as in the corresponding experiments when sheep's



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H. M. VERNON 513

deuterase activated sheep's trypsinogen. In the converse experiment
when sheep's deuterase activated pig's trypsinogen it took, in various experi-
ments, 1-2, 1-3 and 2-3 times longer at 17° than at 37°, whereas we have
seen that pig's deuterase activated pig's trypsinogen as quickly at the lower
temperature as at the higher. Hence the temperature effect depends partly
on enzyme, but probably the physical condition of the substrate is the most
important factor, and it can readily be modified so as to influence the tempera-
ture effect enormously. This is well shown by the two series of curves
reproduced in Fig. 11. In one experiment, the results of which are represented
by continuous line curves, some of the same glycerin extract of pig's pancreas,
which on fivefold dilution with 0-05 % NagCOg was activated as quickly
at 17° as at 37°, was diluted fivefold with water only. It was activated
hy 5 % ot active pig's pancreas extract, and we see that its activation was
distinctly slower at 17° than at 37°, and very much slower at 1°. In the other
experiment the same extract was diluted with an equal volume of water
only, and then it took four times longer to activate at 17° than at 37°, and
much longer still at 1°. The curve obtained at 17° happens to be identical
in position with the 1° curve of the more diluted extract.

Experiments with the active HCl extract of pig's pancreas previously
mentioned gave similar results. When allowed to activate glycerin extract
of pig's pancreas it took 1*6 times longer at the lower temperature, and
when activating sheep's extract it took, in various experiments made at
different times with different amounts of enzyme, 2-2, 2-7, 2-8, 3-8 and 64
times longer at the lower temperature.

The Rate of Liberation of Deuterase.

We have seen that the addition of small quantities of active pancreatic
extracts to inactive extracts rapidly activates the trypsinogen, but if a sample
of this freshly activated extract be added to more inactive extract it is found
to possess little or no activating power. For instance, in Fig. 12 we see
the effect of adding 3 % of active extract of pig's pancreas to inactive extract
at 17°. Activation was almost complete in 30 minutes, and a minute after
this time (marked by an arrow in the figure) a sample of the mixture containing
3 % of the extract was added to more inactive extract at 17°, and we see
from the lowest curve in the figure that it had extremely little activating
power. Another sample of the mixture taken 48 minutes after had distinctly
more activating power : one taken 115 minutes after much more activating

Biocb. vm 34



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514



tt M. VERNON



power still, whilst a sample taken 175 minutes after had almost as much
activating power a£[ the original pancreatic extract. It is concluded, therefore,
that a freshly activated extract contains scarcely any deuterase, but that
this enzyme is gradually liberated from the pro-deuterase present in the
extract by the action of the free trypsin. In proof of this hypothesis, it is
found that the rate of development of activating power is dependent on
temperature. On repeating the experiment at 1°, it was found that 3 %
of the active extract activated the inactive extract to about the same point
as that indicated in Fig. 11 in 90 minutes. At times ranging from 145 to
696 minutes later, 3 % samples of it were added to inactive extract at 17°,




1 min. after



20 50

TIML IN M/NUTCS



40



Fig. 12.

and it was found that the activation curves were of the same character as
those shown in Fig. 11, except that the rate of generation of activating
power in the mixture — which was kept throughout at 1° — was about four
times slower than before. This is shown by the curves given in Fig. 13,
which indicate the percentage of trypsin liberated in 40 minutes on addition
of 3 % samples of the regenerating mixtures to inactive extract. When
the results recorded in Fig. 12 are plotted out in this manner, it is seen that
the rate of development of activating power gets more and more rapid from
start to finish. The results obtained at 1° are plotted out on a time scale
reduced fourfold, and we see that the curve obtained is of similar form and
position to that obtained at 17° ; i.e. that the rate of development of activating



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H. M. VERNON



515



power in freshly activated extracts is four times slower at I*' than at 17°.
At 37° it is probably about four times quicker than at 17°, but the results
are unsatisfactory owing to the extreme instability of the liberated deuterase
at this temperature. Thus an experiment was made in which 3 % of active
extract was added to inactive extract at 37°, and it activated it almost com-
pletely in 24 minutes. At times ranging from 5 to 139 minutes later 3 %
samples of this mixture — ^kept throughout at 37° — were added to inactive
extract at 17°, ^and the percentages of trypsin liberated in 40 minutes are
shown in Fig. 13. We see that though the activating power of the mixture



100



0'2S



7. OF ACTIVE PANCREAS EYT
O'S 1 2

-I-




100
TIML IN MINUTES

Fig. id.



1^0 at J ;• 200
and 3 7*



at first developed much more rapidly than that kept at 17°, it quickly slowed
down, and after 105 minutes began to deteriorate.

The rapidly accelerating rate of formation of deuterase in freshly activated
extracts is a mysterious and unexpected phenomenon. It is true that
within certain limits the amount of trypsin liberated in an inactive extract
on the addition of active extracts increases in greater proportion than corre-
sponds to the extract added. For instance, the curves in Fig. 6 indicate
that the addition of 0-25, 0-5, 1 and 2 % of active extract to inactive extract
caused the liberation, in 40 minutes, of 12, 28, 79 and 193 units of trypsin
respectively, or in the proportion of 1, 2-3, 6-6 and 16-1, instead of 1, 2, 4

34—2



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51G H. M, VERNON

and 8 : but the rate of acceleration of deuterase formation is much more
rapid than this. It occurs in geometrical progression, as is shown by com-
paring the curves of deuterase formation in Fig. 13 with the dotted line
curve. This curve indicates the percentage of trypsin liberated by 0*26
to 4 % of active extract (cf. Fig. 6), but plotted out on the logarithmic scale
given at the top of the figure. The curve is very similar in form to those
indicating the rate of formation of deuterase at 17° and 37°, and hence it
follows that this likewise occurs in geometrical progression.'

In contradistinction to this geometrical increase in the rate of deuterase
liberation is the fact that on addition of small quantities of deuterase to
inactive extracts a certain amount of trypsinogen is activated almost at
once, but then the activation slows down and continues so slowly as to be
incomplete even 24 hours later {cf. Fig. 6). One would have thought that
the trypsin liberated by the deuterase added would at once begin to liberate
more deuterase from the pro-deuterase present, and that in consequence
the activation would soon begin to accelerate again as it does in the presence
of enterokinase. So far from that, the results recorded in Fig. 6 indicate
that 0-5 % of deuterase, though it liberated 26 units of trypsin in 40 minutes,
liberated only an extra 42 units in 22 hours.. Again, 1 % of deuterase
liberated 78 units in 40 minutes and an extra 90 units in 22 hours, though
at the end of this time 52 units of trypsin still remained in the zymogen
form. Experiments with sheep's pancreas extracts showed that the activa-
tion induced by deuterase continued much more steadily than that in pig's
pancreas extracts, but even in them no acceleration was observed. The
explanation of this result is unknown, but it seems to be dependent on condi-
tions, such as the presence of anti-bodies, which do not obtain in pancreatic
juice. As already pointed out, the results recorded in Fig. 9 clearly indicate
that the addition of deuterase to pancreatic juice causes not only a rapid
initial activation of a portion of the trypsinogen, but after an interval of
slow activation this is succeeded by a second acceleration which must be
due to the liberation of fresh deuterase. The results obtained in another
experiment at 17° are recorded in Fig. 14 (see below), and two of the curves
there reproduced show this secondary acceleration. In a similar experiment
carried out at 37° the secondary acceleration appeared in every case.

It is evident that if time be given for the deuterase in freshly activated
extracts to be liberated, the capacity for activation can be transmitted
from extract to extract indefinitely. For instance, 5 % of an active extract
of pig's pancreas was used to activate inactive extract of pig's pancreas,



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a M. VERNON



617



and 4:'5 hours later 5 % of this mixture was added to more inactive extract.
15 hours later 5 % of this mixture was used to activate still more inactive
extract, and 5*5 hours later 5 % of this last mixture was added to still another
sample of inactive extract. All the activation curves in the series were
practically the same in form.

Experiments with glycerin extracts of sheep's pancreas gave results
similar to those obtained with extracts of pig's pancreas. For instance,
5 % of active sheep's extract was added to inactive extract at 17°, and
36 minutes later the addition of 5 % of this mixture to more inactive she^sp's



250-



200



150



100



50-




6 mm. after



W 20 30

TIMC tN MINUTCS



40



50



Fig. 14.



extract at 17° liberated 8 units of trypsin ; 51 minutes later it liberated
13 units ; 150 minutes later 24 units, but 343 minutes later, 165 units. Hence
there was a rapid acceleration in the rate of deuterase formation in the latter
part of the experiment, as in the previous series of experiments. The activa-
tion curves were somewhat similar in form to those shown in Fig. 12, and
were not linear like the sheep's pancreas curves shown in Fig. 7.

Pancreatic juice yielded similar results to pancreatic extracts. In an
experiment at 17°, 3 % of active extract of pig's pancreas activated the
juice completely in 47 minutes, and 6 minutes later 5 % of the active juice
was added to inactive juice at 17°, but as can be seen in Fig. 14 its activating
power was but small. When it was tested again 98 minutes after it had
probably reached its maximum activating power, for when tested 142 minutes



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618 H. M. VERNON

after it was distinctly less active. When tested 5 hours and again 21 hoius
after it had less activating power still. Hence the curves shown in the
figure seem to indicate that the activating power of freshly activated pan-
creatic juice reaches its maximum two to four times more quickly than
in pancreatic extracts, and that the deuterase formed is so unstable as to
undergo rapid destruction even at 17°. In a similar experiment at 37°
3 % of active extract activated the juice completely in 13 minutes, and the
immediate addition of 5 % of this active juice to inactive juice at 37° had
as much activating effect as that shown in the lowest curve in Fig. 14. A
sample taken 25 minutes later had nearly as much effect as that shown
in the " 142 minutes after " curve of Fig. 14, whilst a sample taken 104
minutes after had slightly less effect than that shown in the lowest curve
of Fig. 14. Another experiment at 37° showed that the freshly activated
juice had more activating power 14 minutes after it had completed activation
than either immediately after or 40 minutes after. Hence it is evident that
at 37° the deuterase of freshly activated juice is extraordinarily unstable.
It is quickly liberated, but within a very few minutes most of it is destroyed.

A Comparison between the Activating and Tryptic
Powers of Extracts.

The experimental results thus far described are explicable on the hypo-
thesis which I put forward in previous papers to the effect that trypsin
itself is a powerful activator of trypsinogen. I found that fresh pancreatic
extracts, rich in trypsin, had relatively much greater activating power than
extracts which had deteriorated to some extent in tryptic power as the result
of being kept. Also I found that the trypsin (or rennin) in freshly prepared
extracts is so unstable that even when kept with water only (and no NajCOj)
at 38° about 50 % of it is destroyed in an hour. That in kept extracts
is much more stable, and may be destroyed at the rate of only 12 % per
hour [Vernon, 1901 , 1 , p. 196]. Hence I concluded that trypsin exists in various
states of stability, and that only unstable trypsin has much activating power.
It was suggested to me by Prof. W. M. Bayliss that I had no good evidence
that the activating substance was this unstable trypsin, and further experi-
ments supported his contention, for I found that in various freshly prepared
active pancreatic extracts there was no relationship between activating
power, trypsin content and stability of trypsin. It is quite true that if any
extract deteriorates as the result of being kept, its activating power diminishes



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H. M. VERNON 619

much more rapidly than its tryptic power, but this is because the deuterase
it contains is much more unstable than the trypsin.

Anjrthing more than a rough comparison between the activating powers
of extracts is useless, in that such comparisons do not hold at all closely
at different concentrations. A comparison of the activation curves obtained
with pig's pancreas extracts and sheep's pancreas extracts which are shown
in Figs. 6 and 7 proves this. In experiments made at 37° it was found that
3 % of active sheep's extract activated inactive sheep's extract at the same
rate as 1-25 % of active pig's extract, or that its activating power was 42 %
as great. A similar experiment at IJ"^ showed it to be 40 % as great. In
other experiments made at different times upon inactive pig's extract it
was found that 6, 4*5 and 3 % of active sheep's extract activated at the
same rate as 2-0, 1-5 and 0-65 % of active pig's extract, or was 33, 33 and
22 % as active in the respective experiments. On an average, therefore,
the sheep's extract had only 34 % of the activating power of the pig's extract,
in spite of the fact that its tryptic power was almost exactly the same. The
HCl extract of pig's pancreas, though it had three-fourths the tr}rptic power
of the glycerin extract, had only a sixth its activating power, whilst a watery
extract of pig's pancreas (made by extracting for 42 hours with 2 parts of
chloroform water, filtering off and keeping with chloroform) had about half
the tryptic power of the glycerin extract, but only a thirtieth the activating
power. These experiments with pig's pancreas extracts confirm those formerly
made by me [1901, 2, p. 295], in which I found that 0-9 % NaQ extracts
of the pancreas of various animals had much less activating power, relative
to their trypsin content, than their glycerin extracts.

The most striking evidence of the lack of correspondence between activating
power and tryptic power was obtained with the other two extracts recorded











% Trypsin










destroyed in




Pancreaiio Extract


Relative activating power
Mean


Trypsin


1 hour at 37^


Glycerin


extract of pig


100


294


12





„ „ monkey


83, 62, 62 66


B3


16





„ sheep


42, 40, 33, 33, 22 34


293


65


Ha


» pig


17


218


17


H,0


" P»g


3-5


144


-—


Glycerin


„ ox


1


183


78



in the table. A glycerin extract of monkey's pancreas was found to have
only about a fourth as much tryptic power as the glycerin extract of pig's
pancreas, but two-thirds its activating power. This relative richness of



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520 H. M. VERNON

the monkey's gland in pro-deuterase may help to explain the fact that both
the concentrated glycerin extracts made with the pancreas of healthy Macaque
monkeys, which had been killed instantaneously, activated themselves
spontaneously within a fortnight of their preparation.

In contradistinction to monkey's pancreas extract, a 75 % glycerin
extract of ox-pancreas, though it had nearly two-thirds the tryptic power
of the pig's extract, had no activating power at all in most experiments.
As there was a slight indication of it in one or two instances, its actual power
is recorded in the table as 1 % on that of pig's extract. As will be shown
in the next section, the extract did contain some deuterase, but its activating
powers were masked by the presence of an anti-body.

The stability of the trypsin in pancreatic extracts was tested by diluting
them fivefold with water and NagCOg to 0*05 %, and keeping for an hour
(or two hours) at 37°. Under such conditions no less than 78 % of the trypsin
of the ox-pancreas extract was destroyed, and 65 % of that in the sheep's
extract. The other extracts were much more stable, and the glycerin extract







Extract


% trypsin destroyed in 1 hr. at 37°


Mean


Glycerin


extract of ox pancreas


82, 76, 76


78%


»»





„ sheep's „


71, 70, 70, 69, 64, 69, 62


66


HCl





» pig's „


21, 19, 17, 12


17


Glycerin


»»


„ pig*8 „


27, 26, 23, 23, 22, 21, 21, U (two hours)


(22)


it





„ monkey's pancreas 17, 16


16



of pig's pancreas had only 22 % of its trypsin destroyed in tvH) hours. The
stability of the trypsin probably depends on its colloidal state, and so varies
according to the manner in which an extract of a gland is prepared, for my
earlier experiments with pig's pancreas extracts gave a very different result
from the present ones. When tested by a fibrin digestion method it was
found that the trypsin of a glycerin extract was destroyed at the average
rate of 69 % per hour by 0-4 % NaaCOg at 38°, and at the rate of 39, 32,
24 and 23 % per hour by water only (in this case tested by the metacasein
method). That is to say, it was destroyed about three times more rapidly
than in the present experiments, if we assume that 0'05 % NagCOg is rather
more injurious than water only. A glycerin extract of sheep's pancreas
was destroyed at the average rate of 80 % per hour by 04 % NajCOj, and
at the rate of 66, 60, 53 and 39 % per hour by water only, or was about as
unstable as in the present experiments.

The great instability of deuterase as compared with trypsin is proved
by several experiments described in the previous section, in which the activa-
ting power of juice kept at 17° and 37° was found to deteriorate rapidly.



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H. M. VERNON



521



though the tryptic power was almost unaffected at 17°, and only
moderately affected at 37°. Also I showed previously [1913, p. 335]
that pancreatic juice activated at 37° has much less activating power on
inactive juice than if it is activated at 17°. Deuterase retains its activity
almost undiminished for months in glycerin extracts, but is very unstable
in aqueous extracts. This is indicated by the small activating power of the
HCl and aqueous extracts in the present experiments, and of the 0-9 %
NaCl extracts described in my original experiments. Possibly the trypsin
itself attacks it. Arguing from the great retardation to the action of deuterase
exerted by Witte peptone, it seems probable that pro-deuterase is a protein
substance which is first converted into deuterase, and then destroyed, by
the digestive action of trypsin.



130-



50



1/ ^




/






/


/







/
/



10



20



30 40

TIMC IN MINUTES



50



60



10



Fig. 16.



The Augmentor Action of Intestinal Extract on Deuterase.

It was stated in an earlier section that when ox-pancreas extract is acted
on by enterokinase, it shows the typical acceleration in the velocity of the last
half of activation. On an average this latter half took 29 % of the total
activation time, so the acceleration was not marked, but the mere fact that
it exists at all points to the liberation of deuterase in the extract, or is in
apparent contradiction to the result described above. Further experiment
showed clearly that there is a formation of deuterase in the course of activa-
tion. The addition of 10 % of E. to the extract at 17° gave the curve shown
in Fig. 15. Forty-one minutes after the point in this curve marked with an
arrow had been reached, 20 % of the activated mixture (containing 2 % of E.)
was added to more inactive ox-extract together with 8 % of fresh E. That
is to say, the inactive extract was now being activated by 10 % of E., together



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522 H. M. VERNON

with any deuterase which may have been present in 20 % of the previously
activated mixture. The curve obtained shows the rapid initial velocity
of activation followed by a gradual slowing which is the typical deuterase
effect. This is probably followed by a second acceleration of activation,
though by an oversight sufficient experimental data were not obtained
to prove it, but in another experiment made with 10 % of E. + 10 % of
the activated extract 138 minutes after activation, this second quickening
of acceleration is shown. Presumably it is dependent on the enterokinase
present. In another experiment made 76 minutes after activation, with
10 % of E. + 5 % of the activated mixture, the activation is considerably
accelerated, but at a steady rate throughout.

This experiment leaves no doubt that a moderate amount of deuterase
must have been liberated from pro-deuterase in the extract of ox-pancreas
during the course of its activation by enterokinase, but in such a case how
is one to explain the apparent absence of deuterase as shown in the experiments
of the type described in the last section ? The. subject is a difficult and com-
plex one, and I did not attempt to solve it in detail, but the experiment
to be described points to the existence of an anti-body which neutralises
the deuterase unless its action is augmented by the presence of enterokinase.
Some of the extract of ox-pancreas was activated at 17° by the addition
of 5 % of active extract of pig's pancreas, and 8, 56, 128 and 174 minutes
after activation was practically complete 5 % samples of this activated
extract were added to more inactive extract, together with 5 % of E. In
every case activation occurred more rapidly than in the control experiment
in which E. alone was added. With the 128 minutes sample it was completed
in 81 minutes instead of the 109 minutes taken in the control experiment,
and with the other samples in 84 to 90 minutes. In the absence of E. the
samples had no activating effect at all.

The augmentor action of intestinal extract upon deuterase was proved
by a number of experiments. For instance. Fig. 16 shows the effect upon
inactive extract of ox-pancreas at 37° of (a) 1-25 % of deuterase (active
glycerin extract of monkey's pancreas) ; (6) 1-25 % of deuterase which
had been diluted tenfold and kept with 10 % of E. at 17° for 2 minutes



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