Published May 20, 1938
TRANSFORMATION OF SWINE PEPSINOGEN INTO
SWINE PEPSIN BY CHICKEN PEPSIN
BY ROGER M. HERRIOTT, QUENTIN R. BARTZ, AND JOHN H. NORTHROP
(From tke Laboratories of Tke Rockefeller Institute for Medical Researck, Princeton,
New Jersey, and tke Institute of Experimental Biology, University of California,
Berkeley, California)
(Accepted for publication, November 19, 1937)
575
The Journal of General Physiology
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Previous work (1) has shown that the transformation of swine pepsinogen to swine pepsin at pH 4.0-5.0 is an autocatalytic reaction; i.e.,
the pepsin forms itself from pepsinogen. It has also been found (2)
that swine pepsin and chicken pepsin are distinct immunologically and
that they differ in addition by the fact that chicken pepsin is much less
sensitive to alkali than swine pepsin. The question arises as to
whether swine pepsinogen activated by chicken pepsin would result
in the formation of chicken pepsin or swine pepsin. It would be
expected that the species specificity of the enzyme was already present
in the inactive precursor and that the formation of the active group
in this inactive molecule would be without effect on the species specificity. If this were the case swine pepsinogen would be transformed
to swine pepsin no matter whether the reaction were catalyzed by
swine pepsin or chicken pepsin and, conversely, chicken pepsinogen
would be transformed to chicken pepsin whether the reaction were
catalyzed by chicken pepsin or swine pepsin.
In order to determine the result of such an experiment swine pepsinogen has been activated by the addition of chicken pepsin and it
has been found that swine pepsin is formed under these conditions.
Similarly, chicken pepsinogen when transformed into the active
enzyme by swine pepsin gives rise to chicken pepsin. In a sense,
therefore, it may be said that the swine pepsin becomes "adapted"
when added to chicken pepsinogen since instead of forming more swine
pepsin, as would be the case were it added to swine pepsinogen, it
now forms chicken pepsin. It has been suggested (3) that the formation of bacteriophage and possibly the viruses is analogous to the for-
Published May 20, 1938
576
TRANSFORMATION OF S W I N E P E P S I N O G E N
marion of active enzymes from their precursors and the results of the
present experiments are formally, at least, analogous to t h e a d a p t a tion of bacteriophage to a different host organism.
E X P E R I M E N T A L RESULTS
T h e result of the experiment in which swine pepsinogen was act i v a t e d b y chicken or swine pepsin is shown in Fig. 1 in which the
increase of t o t a l pepsin and of chicken pepsin is plotted against the
time of activation. T h e results show t h a t the pepsinogen solution
to which chicken or swine pepsin has been added activates m u c h
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If an alkaline solution of pepsinogen is brought to pH 4.6 it is slowly transformed into active pepsin and the course of the reaction in general follows that of
an autocatalytic reaction. The reaction is presumably initiated by traces of pepsin already present since it has not been possible to prepare pepsinogen completely
free of pepsin activity. In order to determine the effect of the addition of pepsin
upon the rate of activation, therefore, it was necessary to compare the rate of
activation of the pepsinogen solution alone with that of a solution to which had
been added active pepsin. Owing to the peculiar nature of the autocatalytic
reaction it is necessary to add large amounts of active pepsin in order to markedly
affect the activation curve. The autocatalytic equation predicts that the most
striking difference will be found by comparing the rate of activation of the pepsinogen alone with that of a solution to which has been added about an equivalent
amount of active pepsin. The experiments were therefore carried out by comparing the rate of activation of a solution of pepsinogen which, after activation, would
have an activity of about 1S rennet units per ml., with that of a similar solution
to which had been added sufficient swine or chicken pepsin to bring the initial
activity to 15 rennet units per ml. The total increase in activity of the two
solutions is therefore the same. The experiments were carried out in •/I pH 4.6
acetate buffer at 25°C. Samples were taken into ~/1 pH 5.6 acetate buffer at
various time intervals and also in 0.4 MpH 8.5 borate buffer. In the latter solution
swine pepsin is completely inactivated in about S minutes, whereas chicken pepsin
is not inactivated appreciably for at least ~ hour. 0.5 cc. of the pH 5.6 acetate
samples and also of the pH 8.5 borate samples (after titration to pH 5.0) were
added to 5 cc. of a standard"Klim" (4) solution and the time of dotting determined
at 37°C. The activity, as determined from the acetate sample is called "total
rennet units" and that from the borate sample is called "chicken rennet unit."
One rennet unit is defined as the quantity of enzyme which will clot 10 cc. of a
standard 20 per cent Klim solution in 1 minute at 37°C.
An outline of the method of preparation of chicken pepsinogen is given in Table
I and of chicken pepsin in Table II.
Published May 20, 1938
R. M. HERRIOTT, Q. R. BARTZ, AND J. H. NORTHROP
577
more rapidly than the pepsinogen solution alone and also that the
increase in activity is entirely swine pepsin and that no new chicken
pepsin is formed.
In Fig. 2 the results have been plotted as the log of Ae -- A against
A
the time where A e is the final activity and A is the activity at time T.
This method of plotting gives a straight line for the two experiments
o Swine pepsinogen
Total rennet
z~ Swine pepsinogen+swine pepsin
• Swine pepsinogen+chicken pepsin 6 Swine pepsinoJen
Chicken rennet
2s Swine pepsino~n+swine pepsm
Swine pepsinogen+chicken pepsin
20
*o
0
I0
20
30
40
50
A
;
60
70
Time - minutes
t
80
t
90
6 A ¢
I00
I10
FIG. 1. Effect of the addition of chicken pepsin on the formation of pepsin from
swine pepsinogen.
showing that the reactions are autocatalytic and have about the same
value for the autocatalytic constant. This shows that chicken pepsin
is catalytically as effective as swine pepsin in the activation of swine
pepsinogen. A similar experiment in which chicken pepsinogen was
activated with swine or chicken pepsin is shown in Fig. 3. The results
show again that chicken pepsin is formed from chicken pepsinogen
whether the transformation is brought about by swine pepsin or
chicken pepsin since in this case the increase in activity, from measurements of the samples which had stood at pH 8.5, is the same as that
obtained from the pH 5.6 acetate sample.
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- ,6
Published May 20, 1938
1.2
0.8
0.4
O
o
-0.8
6•
-I.
I
I
I
I
I
I
I
10
20
30
40
50
60
70
I
Time- minutes
Fzc. 2. Activation of pepsinogen shown in Fig. I plotted in accordance with the
.4, - A
autocatalytic equation; i.e., log ~
against the time.
A
tO
o Chicken pepsinogen
Total rennet
• Chicken pepsinogen +swine pepsin
•' Chicken pepsinogen+ chicken pepsin
6 Chicken pepslnogen
Chicken rennet
4 Chicken pepsinogen +swine pepsin
A Chicken pepsinogen + chicken pepsin
.... ÷
E
~ic~,en~r s, ine pepsi%.~
g
.=
m
4
A
6
I0
15
~,~
~
2
0
5
I
20
25
Time-minutes
I
30
I
35
40
FzG. 3. Effect of chicken or swine pepsin on the transformation of chicken
pepsinogen to chicken pepsin.
S78
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-I.2
Published May 20, 1938
R. M. ttERRIOTT~ Q. R. BARTZ, AND ~. H. NORTHROP
~79
1.0
0.8 ~ . =
0.6
0.4
0.2
-0.2
-
-
-0.4 ~ \ .
-~,X~
-0.8
;
\
% ~ =\,,
0
,
I0
~\'l
\
,~
FIG. 4. Activation of pepsinogen shown in Fig. 3 plotted in accordance with
the autocatalytic equation; i.e., log A _ ~ - - against the time.
. t t
I.C
0.4
Oq
0.1
0.2
0.3
I
I
0.4
0.5
06
0.7
MI. serum per 5 ml. milk
I
0.8
I
0.9
&
1.0
FIG. 5. Effect of serum on the rennet activity of various mixtures of swine and
chicken pepsin.
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20 30 40
Time-minutes
Published May 20, 1938
580
TRANSFORMATION OF SWINE PEPSINOGEN
Ae BA
I n Fig. 4 t h e r e s u l t s h a v e b e e n p l o t t e d a g a i n s t t h e log of ----A--a n d a g a i n show fair a g r e e m e n t w i t h t h e course of a n a u t o c a t a l y t i c
reaction.
I n t h i s e x p e r i m e n t t h e final c o n c e n t r a t i o n of p e p s i n was
TABLE I
Outline of Method for Partial Purification of Chicken Pepsinogen
Procedure
Carbohydrate
No. (as glucose)
[P U.IHb*
total rag. lotal 1/mg. N
2
5950
367
0.032
3
1672
330
0.051
4
1080
323
0.065
5
300
262
0.19
7
140
172
0.19
* Activity after transformation into pepsin in acid solution.
half t h a t of t h e first e x p e r i m e n t a n d t h e a u t o c a t a l y t i c c o n s t a n t is
c o r r e s p o n d i n g l y lower.
T h e c o n c l u s i o n t h a t s w i n e p e p s i n is f o r m e d f r o m swine p e p s i n o g e n
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t23 stomachs collected in 0.45 sat. ammonium sulfate - ~r/10
sodium bicarbonate solution. Supernatant discarded . . . .
~tomachs defatted and minced, stirred 12 hrs. with a solution
containing 1102 ml. ~/1 sodium bicarbonate, 1745 ml. sat.
ammonium sulfate, and 8178 ml. water; final concentration
of ammonium sulfate is about 0.2 saturated. 10 per cent
Filter Cel and 5 per cent Hyflo Super Cel added and mixture filtered and washed twice with 0.2 sat. ammonium
sulfate ~t/10 sodium bicarbonate solution. Filtrate refiltered after addition of 1 per cent Filter Cel. Filtrate...
~dded 188 gin. ammonium sulfate per liter. Precipitate
filtered with Hyflo Super Cel and dissolved in 8.5 liters
0.01 u sodium bicarbonate.
¢kddedequal volume sat. ammoniumsulfate dropwise to solution No. 3. Precipitate filtered with aid of Hyflo Super
Cel. Dissolved in 10 volumes 0.01 ~ sodium bicarbonate.
~olution No. 4 made pH 6.0 with 4 ~ pH 4.65 acetate. Added
1.25 volumes of pH 6.0 ~/1 copper hydroxide; filtered.
Precipitate extracted with 3000 nil. ~/10 dipotassium
phosphate. Filtered. Copper hydroxide residue washed
twice with 420 ml. phosphate. Filtrate.
1256gm. ammonium sulfate added to solution No. 5. Precipitate removed by filtration with suction. Cake dissolved in
distilled water, total volume being one-half of No. 4..
~olution No. 6 adjusted to pit 6.0. 1.25 volumes of pH 6.0
u/1 copperhydroxideadded. Filtered. Precipitateextracted with 1500 ml. dipotassium phosphate; filtered. Copper
hydroxide washed twice with 180 ml. phosphate. Filtrate.
Published May 20, 1938
R. M. HERRIOTT, Q. P.. BARTZ, AND J. H. NORTHROP
581
may be confirmed by determining the effect of normal rabbit serum
upon the activated solutions. It has been previously found that
normal rabbit serum inhibits the action of swine pepsin more markedly
than it does chicken pepsin. The results of adding increasing amounts
TABLE II
Outline of Method for Partial Purification of Chicken Pepsin
Procedure
[P.U.IHb
l/mg.N
0.063
0.17
0.17
0.27
0.23
0.29
of normal rabbit serum on the time required for swine pepsin or
chicken pepsin to clot 5 cc. of milk are shown in the two lower curves
of Fig. 5. It may be seen that increasing amounts of serum inhibit
the clotting due to swine pepsin much more than that due to chicken
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398.5 gin. crude pepsinogen (No. 4 of Table I) dissolved in
3,985 ml. 0.01 sodium bicarbonate . . . . . . . . . . . . . . . . . . . . .
Solution No. 1 made pH 6.0 with 4 g pH 4.65 acetate buffer.
Added 1.25 volumes pH 6.0 M/1 copper hydroxide. Filtered. Precipitate extracted with 5100 nil. M/10 dipotassiumphosphate. Filtered. Precipitate washed twice wit[
500 ml. ~r/10 dipotassium phosphate. Filtrate . . . . . . . . . .
314 gin. ammonium sulfate added to each liter of solution
No. 2. Precipitate allowed to settle. Supernatant decanted. 10 gin. Hyflo Super Cel added and precipitate filtered by suction on hardened filter paper. Cake stirred in
2 volumes water. Filtered. Residue washed with 1.5
volumes. Filtrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 volume 0.1 s hydrochloric acid added to solution No. 3~
stood at room temperature for 30 minutes . . . . . . . . . . . . . . .
Adjusted to pH 3.4 with 4 ~t 'acetate buffer. Cooled tc
- 1 0 ° C . Added equal volume cold acetone . . . . . . . . . . . . .
Filtered by suction. Discarded precipitate. Filtrate . . . . . .
10 volumes water added to solution No. 6; 4 gin. Filter Cel
added. After stirring suspension filtered on No. 3 Whatman filter paper by suction. Clear filtrate~. . . . . . . . . . . . .
Solution adjusted to pH 3.0 with N/2 sulfuric acid and equal
volume saturated magnesium solution added, pH readjusted to 3.0 with 5 N sulfuric acid. Stood in cold room
for 2 days. Added 20 gm. Filter Cel. Filtered by suction
on No. 3 Whatman filter paper. Filtrate discarded . . . . . .
Precipitate extracted with N/10 sodium acetate . . . . . . . . . . .
Published May 20, 1938
582
T R A N S F O R M A T I O N OF S W I N E P E P S I N O G E N
pepsin. The two upper curves show the effect of increasing amounts
of serum on the clotting of milk caused by a solution of swine pepsinogen activated by swine pepsin and of a solution of swine pepsinogen
activated with chicken pepsin. If the activation of swine pepsinogen
with an equivalent amount of chicken pepsin results in the formation
of swine pepsin the resulting solution should contain equal amounts of
swine and chicken pepsin. The results show that the inhibiting effect
of serum on swine pepsinogen activated by chicken pepsin is the same
as that on a solution made up by mixing equal amounts of swine
pepsin and chicken pepsin and is less than the effect on the solution
containing only swine pepsin.
SUMMARY
REFERENCES
1. Herriott, R. M., and Northrop, J. H., Science, 1936, 83, 469.
Herriott, R. M., J. Gen. Physiol., 1938,21, 501.
2. Seastone, C. V., and Herriott, R. M., J. Gen. Physiol., 1937,20, 797.
3. Northrop, J. H., J. Gen. Physiol., 1938, 21,335.
4. (Standard Klim) Kunitz, M., J. Gen. Physiol., 1935, 18, 459.
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A~ctivation of swine pepsinogen with chicken pepsin results in the
formation of swine pepsin.
Activation of chicken pepsinogen with swine pepsin results in the
formation of chicken pepsin.
The structure responsible for the species specificity of the enzyme
is therefore present in the inactive precursor.