Tht Journal of Biochemist^. VoL 53, No.

6, 1963
Studies on Snake Venom*
XIII. Chromatographic Separation and Properties of Three Proteinases
from Agkistrodon halys biomhoffii Venom**
By MASATOSHI SATAKE, YORIHIKO MURATA and TOMOJI SUZUKI
(From Dipartmtnt of Biochemistry, Faculty tf Pharmacy, Kyoto University, Kyoto)
(Received for publication, October 25, 1962)
The authors have made a systematic in-
vestigation of the enzymes in the venom of
Agkistrodon halys biomhoffii (Mamushi) which is
a representative of Japanese poisonous snakes.
It was found that the proteinase activity of
Mamushi venom could be separated into
three peaks by means of fractionation on
DEAE-cellulose. It is interesting that several
different enzyme proteins are involved in one
enzymatic activity, although similar pheno-
menon was already reported and discussed
by the present authors (/) and others (2)
on the phosphodiesterase activity of snake
venoms.
As few studies have appeared on the
purified proteinases of snake venoms, it is
believed that a clarification on the character-
istics and specificities of these three proteinases
would be valuable.
Our studies were based on the presump-
tion that a relationship would be demonstra-
ble between the enzymatic activities and the
toxicity of Mamushi venom, especially in
connection with its hemorrhagic and necrotic
activities.
In this paper, the chromatographic sepa-
ration of the three proteinases from Mamushi
XII. Murata, Y., Satake, M., and Suzuki, T.,
/ . Biochm., 53, 431 (1963)
* A part of thu paper wai reported orally at
the 13th Japan Pharmaceutical Assembly (1960)
The abbreviations used were: BAEE, a-benzoyl-
L-arginine ethyl ester; BAA, a-benxoyl-L-argininamide ;
TCA, trichloroacetic acid; CM-, carboxymethyl-;
DEAE-, diethylaminoethyl-; PCMB, />-chloromercury-
benzoate; EDTA, ethylenediamine tetraacetic acid;
ATEE, or-acetyl-L-tyrosine ethyl ester.
venom is described. The differences in their
stability and the inhibitory effects of various
reagents, especially of cysteine, will be pre-
sented. All of these proteinases were inhibited
by EDTA. The proteinases in Mamushi
venom seem to be of a different type from
the known digestive enzymes such as trypsin
[EC 3.4.4.4].
EXPERIMENTAL
Snake Venom and AntiurumThe venom of Japa-
nae Agkistrodon halys biomhoffii (Mamushi), frozen
immediately after collection, was lyophilized and
uied. Mamushi antiserum was a product of Takeda
Pharmaceutical Industries, Ltd., prepared from horse
i:mm immunized only with Mamushi venom. Its
potency was 300 I.V.LD
U
units/mL
SubstraUsCasein according to Hammarsten was
a product of E. Merck tc Co. Crystalline murami-
due [EC 3.2.1.17] was prepared by the method of
Al dert on it al. (5). Examination of its N-terminal
amino acid by the dinitrophenylation method of
Si nge r (4) gave only di-dinitrophenyllysine. BAEE
wu a product of Nutritional Biochemicals Corpora-
tion. ATEE was a product of Tokyo Chemical
Industry Co., Ltd.
Ion Exchange Fibtrt CM-cellulose and DEAE-
cellulose were prepared in this laboratory according
to Pe t e r s o n and Sober (5), with cellulose powder
from Toyo Roshi Kaisha Ltd., Tokyo. Phospho-
cellulose (0.79 meq./g-)
w
& * product of Serva
Entwicklungslabor, Heidelberg.
Salts and Miscellaneous ReagentsMetal salts, such
as MnCl,-4H,O, CaCU,-2H,O, MgCl,-6H,O,
CuSO
4
-5H,O, CoCljoH^O, Zn(NO,),-6H,O, HgClj
and CdClj'H,O were of analytical grade from com-
mercial sources. All other reagents were also of
analytical grade.
Estimation of ProUinsThe protein content in the
438

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Snake Venom Proteinases. XIII
439
cbromatographic fractions was estimated by its UV-
absorption at 280 rapt, based on the absorbancy of
1 mg./ml. solution of crude Mamushi venom being
1.20.
Assay qf Enxjmt AtturityAssays for casein diges-
tion and hydrolysis of amino acid esters were carried
out as described in the previous paper (6). Assays
of chromatographic fractions were made after ap-
propriate dilution of the eluates. For the investiga-
tion of the hydrolysis of muramidase and effect of
pH, the assay procedures were modified
1
. The details
are each described in the footnotes of Figs. 6 and
7. In the investigation of the effects of metal ions
on proteinase activity, high blank color development
in the Folin reaction was observed in the presence
of MB**. TO avoid the error due to this high
blank, casein digestion was estimated by measuring
the absorbancy of the TCA-soluble product at 280 mfi.
The usual Folin's method was used for reference.
Ckromaiagraphic TickniqutsFor the fractionation
of 200 mg. of crude Mamushi venom, a column
(I.3X 16.5cm.) was made with DEAE-cellulosc
(chloride form), and washed with the starting buffer
until no chloride ion was detected in the effluent.
The venom solution (200 mg. dissolved in 2 ml. of
the starting buffer) was applied and gradient elution
was achieved by changing the molarity of Na acetate
buffer, pH 7.0, as follows: The elution was started
with 330 ml. of 0.005 M buffer in the mixing chamber
and 0.1 M buffer in the reservoir, which was changed
with 0.2, 0.4 and 1.0 M buffers at the emergence of
the fractions Nos. 32, 116 and 156, respectively.
Flow rate was adjusted to 911ml. per hour and
6.5 ml. fractions were collected. All procedures were
carried out in the cold room at 4C.
For the fractionation of 1 g. of venom, the
column size was enlarged to 2.2x38 cm. Gradient
elution system was as follow*,: 800ml. of 0.005 M
Na acetate buffer, pH 7.0, was present in the mixing
chamber, and the reservoir contained 0.1 M buffer
which was changed to 0.2, and 1.0 M buffers at the
emergence of franctions Nos. 118 and 246, respect-
ively. Flow rate was 1520 ml. per hour. Each
fraction contained 9 ml.
RESULTS
Chromatography of Mamushi Venom on DEAE-
cdhdost ColumnChromatographic fractiona-
tions of 200 mg and 1 g. of Mamushi venom
on DEAE-cellulose are presented in Figs, la
and lb, respectively. Casein was used as
substrate in tie enzyme assays. The proteinase
activities were separated into three peaks
which were designated in the order of their
elution from the column as proteinase a,
proteinase b and proteinase c. This pattern
was established in several repeated experi-
ments, always giving three proteolytic peaks.
Proteinase a was always found in the first
peak in which the proteins were not adsorbed
by DEAE-cellulose. Proteinase b, however,
emerged occasionally a little faster or later
than the associating peak of protein. Pro-
teinase c was demonstrated always a little
behind the last peak of protein.
The fractions shown in Fig. lb were as-
sayed for BAEE hydrolyzing activity. It
must be noted that the BAEE hydrolyzing
activities were also separated into three peaks,
but only one of them was superimposed with
a proteinase activity (proteinase a). The other
two peaks of BAEE hydrolyzing activity were
distributed in different fractions from those
containing proteinases b or c. A detailed
study on the BAEE hydrolyzing activity will
be reported in another paper.
The following fractions were pooled, con-
QrocQtnt
to<Ui/-+toH/-
60 90 120
TUBE NUMBER
Fio. la. Chromatographic separation of pro-
teinases on DEAE-cellulose column starting from
200 mg. of Mamushi venom.
200 mg. of Mamushi venom was applied on
a DEAE-cellulose column (1.3X 16.5cm.), and
elution was carried out with concentration
gradient of Na acetate buffer, pH 7.0, from
0.005 M to 0.5 Id as described in experimental.
Flow rate, 9-11 ml./hour; collection of effluent,
6.5ml./tube. Proteinase activity was illustrated
by AEgao/15 minutes/0.5 ml. fraction.

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440
M. SATAKE, Y. MURATA and T. SUZUKI
60
eo tso wo aoo
TUBE NUUMR
Fio. lb. Chromatographic separation of pro-
teinases on DEAE-cellulose column starting from
1 g. of Mamujhi venom.
1 g. of Mamushi venom was applied on a
DEAE-cellulose column (2.2x38 cm), and was
eluted with concetration gradient of Na acetate
buffer, pH 7.0, from 0.005 M to 0.5 M as de-
scribed in experimental. Flow rate, 15-20 ml./
hour; collection of effluent, 9 ml./tube. Pro-
teinase activity was expressed by AE^/15
minutes/0.5 ml. fraction.
centratcd by lyophilization, and finally
dialyzcd against distilled water: in Fig. la,
Nos. 28 as proteinase a; Nos. 66120 as
proteinase b; Nos. 134165 as proteinase c:
and in Fig. lb, Nos. 1015 as proteinase a;
Nos. 260290 as proteinase b; Nos. 315345
as proteinase c. These materials were then
subjected to rechromatographies as will be
described later.
The recovery of proteinase activity was
no more than 80 per cent. This was due to
losses on sampling for assays and cutting off
of fractions of low activity. The activities
of the separated proteinases toward several
substrates are shown in Table I. The activi-
ties of the three proteinases toward casein
were 1.21.5 times higher than, that of crude
Mamushi venom, and were 1/151/13 of that
of trypsin. With BAEE, the synthetic sub-
strate of trypsin, proteinasc a was apparently
the most active of the three proteinases, how-
ever, the activity was as low as about 1/30 if
compared with that of trypsin. The activities
of proteinases b and c toward this substrate
were very weak. As was observed with crude
Mamushi venom, each proteinase showed
only slight activity with BAA. ATEE, the
synthetic substrate of chymotrypsin, was
hydrolyzed slightly by proteinases b and c,
but not by proteinase a. Other enzyme
activities that arc commonly found in snake
venoms were also tested with these proteinascs.
Phosphodiesterase [EC 3.1.4.1], 5-nucleotidase
[EC 3.13.5] and phospholipase A [EC 3.1.1.4]
activities were contained in the proteinase a
fraction. Proteinases b and c, however, were
found to be essentially free of these enzyme
activities.
TABLE I
Actuitiis of Thru ProUinuus toward Casrin,
BAA, BAEE, and ATEE
Activities toward casein and BAA were esti-
mated with the enzymes from Fig. la. Activities
toward BAEE and ATEE were estimated with
the enrymes from Fig. lb. Trypsin [EC 3.4.4.4]
and a-chymotrypsin [EC 3.4.4.5] were crystalline
preparations from Nutritional Biochemicalj Cor-
poration.
Proteinase a
Proteinase b
Proteinase c
Trypsin
a-Chymotrypsin
Casein
25.2
29.7
30.4
390
BAA
0.09
0.18
0.09
BAEE
9.2"
4.2
0.5
262
3.0
ATEE
0"
0.5
1.6
0
84
1) fig. tyrosine equivalent of TCA-soluble product/
minute/mg. enzyme.
2) /jmoles NH, liberated/hour/mg. enzyme.
3) ftmoles ester hydrolyzed/10 minutes/mg. enzyme
Further Purification of Protrinast aThe
proteinase a fraction is basic and emerges in
the first peak without being adsorbed by
DEAE-cellulose; therefore, cation exchange
fibers such as CM-cellulosc or phospho-
cellulose can be used as adsorbents to achieve
further purification of proteinase a. In these
chromatographies, the pH of the eluting
buffer was lowered to 5.86.0, instead of the
pH 7.0 buffer used in DEAE-cellulose chro-
matography. The experimental conditions
and results are given in Figs. 2a and 2b. By
these procedures the activity of proteinasc a
toward casein was further separated into two

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Snake Venom Proteinases. XIII 441
,0.3
Qredknt
to 0.28 U
i
40 ISO 80 IfO
TUBE NUMBER
Fio. 2a. Rechromatography of proteinase m
on CM-celluloie.
28.3 mg. of proteinase a recovered in the
fint DEAE-cellulose chromatography wai applied
on a CM-cellulose column (1.3x21.Ocm.) equi-
librated with 0.005 M Na acetate buffer (pH
5.8). Gradient elution wu carried out with
350 ml. of 0.005 M Na acetate (pH 5.8) in the
mixing chamber, and 0.25 M Na acetate (pH
5.8) in the reservoir which wai changed with
0.5 M buffer at the emergence of fraction No.
120. Flow rate was adjusted to 12 ml./hour,
and each fraction contained 6 ml. Proteinase
activity was illustrated by AE^/IS minute>/0.5
ml. fraction.
peaks. Upon CM-cellulose chromatography,
the main peak runs behind the second peak.
In the following experiments the term, "pro-
teinase a" refers to the fraction which is
eluted as the main peak upon CM-cellulose
chromatography. Proteinase a was found to
be accompanied by BAEE hydrolyzing activity
during the chromatographic fractionation.
Richromatographiss of Prottinases b and c
Proteinases b and c obtained after the chro-
matography shown in Fig. la were each sub-
jected to rechromatography on DEAE-cellulose
to ascertain their homogeneity and the relia-
bility of the chromatographic techniques.
The actual conditions and results are given
in Figs. 3 and 4. Upon rechromatography,
the activity of proteinase b emerged in a
single peak and paralleled the UV-absorption
values. The activity of proteinase c, however,
ran a little behind the UV-absorption values,
and showed some inhomogeneity. In any
case, the activities of proteinases b and c emerg-
0.9
J "
to 0.5*
0.9-
|
0.6 J
g
30 ISO
z
I
o E
SO O ISO
TUBE NUMBER
Fio. 2b. Rechromatography of proteinase a
on phospho-celluloie.
173 mg. of proteinase a recovered in the
first DEAE-cellulose chromatography was applied
on a phospho-cellulose column (1.5x22.5 cm.)
equilibrated with 0.005 M Na acetate buffer,
pH 6.0. Gradient elution was carried out with
700 ml. of 0.005 M Na acetate (pH 6.0) in the
mixing chamber, and 0.1 M Na acetate (pH 6.0)
in the reservoir which was changed with 0.5 M
buffer at the emergence of fraction No. 90.
Flow rate was adjusted to 1015 ml./hour, and
each fraction contained 10 ml. Proteinase activity
was illustrated by AE^/IS minutes/0.5 ml.
fraction.
ed in a single peak at approximately equal buf-
fer concentrations required for their elution in
the original chromatography, thus proteinases
b and c were individual enzymes and the
separation of the proteinases in the original
chromatography was not an artifact.
Anligtn-antibody RtactionAntigen-antibody
reactions were tested according to the semi-
solid precipitin methon of B o w c n (7). From
our previous study on the detection of the
common antigens reacting with Mamushi
antiserum among Japanese and Formosan
snake venoms, we knew of the presence of at
least seven antigens in crude Mamushi venom
(ff). In a small test tube (4x70 mm.) were
placed antiserum diluted fivefold in 0.4%
agar in the bottom layer (10 mm. in height),
an intermediate 0.8% agar layer (30 mm.),
and the proteinase solution in the top layer
(25 mm.). The appearance of the precipitation
lines in the intermediate layer by the reac-
tions between the diffused reactants was

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442 M. SATAKE, Y. MURATA and T. SUZUKI
O 20 40 80 80 100
TUBE NUMBER
Fio. 3. Rechromatography of proteinaie b
on DEAE-cellulosc.
29.5 mg. of proteinase b recovered in Fig.
la was applied on a DEAE-cellulose column
(1.3x9.0 cm.) equilibrated with the starting buf-
fer. Gradient elution was carried out with 300
ml. of 0.05 M acetate (pH 7.0) set in the mixing
chamber, and 0.5 M acetate of the tame pH in
the reservoir. Flow rate was 710 ml./hour,
and fractions were collected by 5.0 ml./tube.
Proteinase activity was illustrated by
minutes/0.5 ml. fraction.
observed in the course of a week's incubation
at 37C. In these experiments, three indistinct
zones of reaction appeared in the tube con-
taining proteinase a, indicating considerable
inhomogeneity of proteinase a. This is in
agreement with the result of rechromato-
graphy of proteinase a on CM-cellulose, in
which the UV-absorbing material was broadly
distributed in the main peak of proteinase a-
Proteinase b, which was eluted in a homo-
geneous single peak, gave only one distinct
line of reaction. Proteinase c was eluted in
a single peak but did not parallel the absor-
bancy values on rechromatography, and in
accordance with this result proteinase c gave
two distinct lines in the antigen-antibody
reaction.
Exhaustive Hydrolysis of Cas tin and Murarrd-
dase by Protsinases a, b, and c-.We have routine-
ly employed the hydrolysis of casein during
incubation for 15 minutes as a measure of
proteinase activity. However, in the hope
that we may ascertain differences in the
character of these proteinases, each enzyme
40 80 80 100
TUBE NUMBER
Fio. 4. Rechromatography of proteinase c
on DEAE-cellulose.
31.1 mg. of proteinase c recovered in Fig.
la was applied on a DEAE-cellulose column
(1.3x8.0 cm.) equilibrated with the starting
buffer. Gradient elution was carried out with
250 mL of 0.1 M acetate (pH 7.0) set in the
mixing chamber, and 0.5 M acetate of the same
pH in the reiervoir. Flow rate was 7.59.5
ml./hour, and fractions were collected by 5.5
ml./tube. Proteinase activity was illustrated by
minutes/0.5 ml. fraction.
Fio. 5. Exhaustive hydrolysis of casein by
venom proteinases.
4.0 ml. of 2% casein solution (pH 8.5) wat
incubated at 37C with 4.0 mL of enzyme solu-
tion containing one of the following proteinases;
810/tg. of proteinase a, 690pg. of proteinase b,
or 790 ftg. of proteinase c. At time intervals
ihown in the figure, 1.0 ml. aliquot was taken
out and added into 1.0 ml. of 0.44 M TCA.
Then the acid-soluble material was estimated in
the usual method.
: proteinase a, O: proteinase b,
X : proteinase c.

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Snake Venom Proteinases. XIII 443
was incubated for 12 hours with casein and
the increases in TCA-soluble materials were
determined at the intervals shown in Fig. 5.
The extent of hydrolysis of casein by pro-
teinase b was apparently twice as great as
those obtained with proteinases a and c. The
difference observed in the degree of casein
digestion by these proteinases indicates the
possibility of characterizing these enzymes by
the use of different substrates. Accordingly, pro-
longed digestion was investigated under more
rigid conditions with another substrate, heat-
denatured crystalline muramidase (Fig. 6).
In contrast to the findings obtained with
casein, the use of muramidase resulted in the
highest activity with proteinase a. The libera-
tion of TCA-soluble material was greatest
with proteinase a, followed by b and c, re-
spectively. The release of TCA-soluble mate-
rial from denatured muramidase ceased after
6 hours in tubes containing proteinases b and
24
Fio. 6. Hydrolysis of crystalline muramidase
by venom proteinases.
1.5 ml. of 1% muramidase solution was in-
cubated at 37C with 0.5ml. of enzyme solution
containing one of the following proteinases;
1.28 mg. of proteinase a, 2.21 rag. of proteinase
b or 1.80 mg. of proteinase c. At the time in-
tervals shown in the figure, 0.2 ml. aliquot was
taken out and added into 1.0 ml. of 0.44 M TCA.
After standing for 30 minutes, the mixture was
filtered and 0.2 ml. of the filtrate was taken for
measurement of acid-soluble material by Folin
reaction. Muramidase solution was made by
dissolving crystalline muramidase at 1% in water,
adjusting to pH 7.0 and finally heating at 100C
for 6 minutes.
#: proteinase a, O: proteinase b,
X: proteinase c.
c, whereas with proteinase a, slow liberation
continued for at least 12 hourse.
Optimum pHThe effect of pH on the
proteinase activity toward casein is shown
in Fig. 7. Each of the proteinases was active
in the alkaline pH region, showing different
pH optima; the values were 10.5, 9.8, and 8.9
for proteinases a, b, and c, respectively.
100 -
Fio. 7. Effect of pH on venom proteinase*.
Reaction mixture contained 0.5 ml. of
. enzyme solution and and 0.5 ml. of 2% casein
at various pH. The activities were illustrated
in terms of relative activity to the maximum
activity. The substrate solutions were made as
follows: Casein was dissolved at 8% in 0.4 M
Tris-HCl buffer of pH varying from 7.2 to 9.0,
and heated at 100C for 15 minutes, then diluted
with water to make 2% as regards with casein.
To make casein solution at pH below 7.0 or above
9.0, the pH was brought to desired value by adding
0.2 N HC1 or 0.2 N NaOH before dilution. All
casein solutions were checked for their pH before
use.
: proteinase a, O: proteinase b,
X : proteinase c.
StabilityDifferences in heat stability
among proteinases a, b and c were revealed
by heating for 10 minutes in the range of
4080C as shown in Fig. 8. The results
indicated that proteinase c is the least stable
of the three. The activity of proteinase c is
almost completely destroyed (90%) by heating
at 60C- Proteinase b was 70% inactivated,
whereas 80% of proteinase a activity remained
under the same condition. All of the pro-
teinases were inactivated completely by heat-
ing for 10 minutes at 80C.

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444
M. SATAKE, Y. MURATA and T. SUZUKI
IOO
40 DO 60 70
TEMPtRATURE CO
SO
Fio. 8. Heat stability of venom proteicasei.
0.5 ml. of enzyme solution was heated at
indicated temperature for 10 minutes in a water
bath and immediately cooled. Then the solutions
were assayed in the usual manner. Incubation
was at 37*C for 10 minutes. The concentration
of enzyme solutions were: proteinase a, O.M
mg./ml.; proteinase b, 0.79 rag./ml.; proteinase
c, 0.73 mg./ml. The activities were illustrated
in terms of relative activity to that of unheated
control enzyme.
: proteinase a, O: proteinase b,
X : proteinase c.
Effict ofMitd IonsThe effects of divalent
metal ions on the proteinasc activities were
investigated. Ca
++
and Mg
++
were slightly
stimulatory. All other divalent metal ions
acted as inhibitors. Cd
++
, particularly, had
a strong inhibitory effect (Table II).
Effects of Otfur Ag$ntsThe proteinase
activities were strongly inhibited by EDTA,
in agreement with the results of others (9
IT). Proteinase c was inhibited completely by
2x 10"* M EDTA. PCMB, a sulfhydryl group
bloclung agent, was only slightly inhibitory.
On the other hand, SH-componds such as
glutathione and cysteine also seem to inhibit
the proteinases (Table III). The effect of
pretreatment of the enzymes with cysteinc,
as shown in Table IV, revealed distinct
differences among three proteinases. Protein-
ase c was inactivated completely by treatment
for 30 minutes with 4x10"' M cysteine.
Efftct of AntistnmIt has been pointed
out in the previous study that Mamushi
venom antiserum contains several anti-en-
TABLE II
Sjfftct of Mttal Ions on tht Pnttixaa Acturitus
0.8 ml. of enzyme solution in 0.1 M Trii-
HC1 buffer (pH 8.5) was mixed with 0.2 ml. of
2xlO~* M metal solution, and incubated for 5
minutes, lml. of 1% casein in 0.1 M Trij-HQ
buffer (pH 8.5) was added and incubated for 20
minutes at 17*0. Then 3 ml. of 5% TCA was
added, and the mixture was centrifuged after
standing for 1 hour. The TCA-soluble product
was determined by measuring the absorbancy at
280 m/L The values were expressed in terms of
the relative percentage of activity to the control
system (no addition of metal salt). The actual
values of AEgo in the control system were as
followi: 0.209 for proteinase a; 0.219 for pro-
teinase b; and 0.250 for proteinase c.
Addition
None
CaCl,
MgCl,
Mn(3,
CuSO
4
Znr.NO,),
CoCl,
HgCl,.
CdCl,
Relative activity
Proteinase a
100
117
120
48
43
33
14
7
0
Proteinase b
100
106
102
23
16
26
7
7
1
Proteinase c
100
133
116
24
16
27
20
11
0
zymes against the enzymes in Mamushi
venom. The effects of antiserum on the three
proteinases are shown in Table V. It was
found that inhibition by antiserum was
observed with each of the proteinases. The
degree of inhibition depended on the con-
centration of antiserum, but very high con-
centrations of antiserum were required for
the effective inhibition of the proteinases.
In a previous study, up to 80-fold dilution
of antiserum had been demonstrated to retain
effective anti-enzyme activity against 5-nucle-
otidase and phosphomonoestcrasc activities
(#). However, with the proteinases, even a
25-fold dilution was found to cause ineffective
inhibition of proteinase activities. This was
especially marked in the case of proteinase b
(Table V).

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Snake Venom Proteinases. XIII 445
TABUS i n
Efftcls qf Various Inhibitors on Vtnom ProUmau Actwtius
Reaction mixtures contained 0.5 mL of 2% casein solution, 0.3 ml. of enzyme solution and 0.2 ml.
of inhibitor solution (1x10"' M) or water. The final inhibitor concentration was 2 X 10-* M. Incubation
was carried out at 37*C for 60 minutes with proteinase a, 20 minutes with proteinase b, and 30 minutes
with proteinase c, respectively. The values were expressed in terms of the relative percentage of acti-
vity to the control system (no addition of inhibitor). The actual values of activities (AEm) in the
control system were as follows: 0.108 for proteinase a; 0.455 for proteinase b; and 0.313 for proteinase c.
None
PCMB
EDTA
Thioglycolate
Glutathione
Cystcine
Proteinase a
100
94 .
17
79
29
44
Relative activity
Proteinase b
100
81
16
97
82
90
Proteinase c
100
100
3
103
27
34
TABLE IV
Efftct of Cjsttvu on Vtnom HoUaau Attimtut
0.3 ml. of enryme solution was pretreated with 0.2 ml. of lxl0~* M cyiteine or 0.2 mL of water at
37C for 30 or 60 minutes, then 0.5 ml. of 2% casein solution was added and the activity during 30
minutes incubation was measured by the routine method. The enzyme concentrations were as follows:
proteinase a, 200pg./ml.; proteinase b, 290/ig./ml.; proteinase c, 275/./ml. Suitable correction was
made for the blank color development due to the coexistence of cyiteine.
Pretreatment
Addition j
Water :
Cysteine j
Cysteine
Time
30 minutes
30 minutes -
60 minutes
Proteinase a
0.260
0.106
0.075
Activity (AE^j/30 minutes)
! Proteinase b
i
0.360
0.220
0.210
Proteinase c
0.340
0
0
DISCUSSION
It has been shown by several authors that
snake venom contains several enzymes con-
cerned with one enzyme activity. Three
phosphodiesterases have also been separated
in this laboratory from Mamushi venom.
Maeno et al. (9). and Ohsaka (10) have
reported independently the presence of two
or more proteinases in Trinunsurus venom.
It was shown in this work that Agkistrodon
halys blomkoffii venom contains three or more
proteinases. The three proteinases were
obviously of different characters in view of
the difference in pH optima, heat stability
and the effect of cysteine. An important
finding is that proteinases b and c were
separable from BAEE hydrolyzing activity.
Hitherto, in experiments with crude venom,
it had been believed that the proteinase of
snake venom possesses BAEE hydrolyzing
activity, and it was one of the bases which
make the proteinase in snake venom to be of
trypsin-type (12), in addition to their activity
toward casein and hemoglobin and its pH
optima. It must be recalled that Ha mbe r g
tt al. (IS) have demonstrated a difference in
heat stability between the activities toward

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446 M. SATAKE, Y. MURATA and T. SUZUKI
TABLB V
Inhibition qf Mamushi Vtnem Pnttixau by Mamushi Antistrum
0.3 ml. enzyme solution and 0.2 ml. of antuerum solution diluted adequately with 0.9% NaCl were
mixed to make indicated concentration as regards with antiierum, and kept at 37C for 5 minutes, then
0.5 ml. of 2% casein solution wai added. The incubation periods for the assays of remaining proteinase
activity were 60 minutes with proteinase a, or 30 minutes with proteinases b and c. The values were
expressed in terms of the relative percentage of activity to that of the control system. The actual
values of activities (AEm) in the control system were as follows: 0.120 for proteinase a, 0.580 for
proteinase b, and 0.340 for proteinase c.
Control
X 50
X 25
X 12.5
X 5
Proteinase a
100
48
19
8
6
Relative activity
Proteinase b
100
94
85
68
29
Proteinase c
100
49
12
5
2
casein and benzoylarginine methyl ester, and
have considered that the proteinase and
esterase may be two different enzymes. The
proteinase and BAEE hydrolyzing activities
may be represented by different enzymes on
the basis of the result of chromatographic
studies and previous observation with ten
kinds of venoms (5). Furthermore, protein-
ases a, b and c have different activities as
revealed by exhaustive hydrolysis experiments.
From the results of the hydrolysis of casein
and crystalline muramidase, it is anticipated
that proteinases a, b and c will show different
specificities toward the peptide bonds to be
split. Further studies are now in progress on
the specificities of these proteinases using B
chain of insulin and glucagon. While we
were employed in this specificity studies,
Pf l e i de r e r it al. {II) published the separa-
tion of three proteinases from Crotalus atrox
venom- Interestingly, their enzymes have
similarity to Mamushi venom proteinases in
the peptide pattern of the digest of insulin
B chain. Studies on the substrate specificities
of three proteinases of Mamushi venom will
be presented in the following paper.
SUMMARY
The venom of Ag/nstrodm halyt blomhoffti
(Mamushi) contains three proteinases. These
proteinascs could be separated on DEAE-
cellulose, and were designated as proteinases
a, b and c These proteinases have different
pH optima: the values were 10.5, 9.8 and 8.9
for. proteinases a, b and c, respectively. They
showed different stability against heating or
treatment with cysteine, and proteinase c was
the most unstable. These proteinases were
activated by Ca
++
and Mg
++
, but were inhi-
bited by other divalent metal ions. All of
these were inhibited by EDTA. From the
results of exhaustive digestion of casein and
muramidase it was considered that these
proteinases have different substrate specifici-
ties from each other. During the chromato-
graphic fractionation of these proteinases,
BAEE hydrolyzing activity was found to be
separable from proteinase activity, at least
from proteinases b and c
The authors wish to express their thanks to Mr.
S. Kawachi of Hoshi College of Pharmacy for his
valuable help in this work. Thanks are also due to
Dr. S. Iwanaga, T. Omori, T. Sato, and Y. Mizu-
shima of this laboratory for their help in this work.
This work was supported, in part, by a grant for
scientific researches from the Ministry of Education
for " Enzymatic studies on animal toxins," to which
our thanks are due.
REFERENCES
( / ) Suzuki, T., Iwanaga, S., and Satake, M., ],

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Snake Venom Protcinases. XIII 447
Pharm. Soc. Japan (in Japanese), 80, 857, 861
(1960); Suzuki, T., Iwanaga, S., and Nitta, K...
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(2) Boman, H. G., and Kaletta, U., Biochim. it
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(3) Alderton, G., Ward, H., and Fevold, H. L.,
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(6") Murata, Y., Satake, M., and Suzuki, T., / .
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( 7) Bowen, H. E., / . Immunol., 68, 429 (1952)
(tf) Iizuka, K., Murata, Y., and Satake, M., / .
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( 9) Maeno, H., Miuuhashi, S., and Sato, R.,
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(JO) Obsaka, A., Japantsi J. Mid. Sd and Biol., 13,
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(11) Pfleiderer, G., and Sumyk, G., Biochim. it
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(12) Kaiser, E., and Michl, H., "Die Biochemie
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(13) Hamberg, TJ., and Rocba e Silva, M., Arch,
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