Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963

8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963

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Th tJournal

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 forpublication, October 25, 1962)

The authors have made a systematic in-

vestigation of the enzymes in thevenomof

Agkistrodon halys biomhoffii (Mamushi) which

is

a representativeofJapanese poisonous snakes.

It was found that the proteinase activityof

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 inone

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 clarificationon thecharacter-

istics and specificitiesofthese three proteinases

would bevaluable.

Our studies were basedon thepresump-

tion that a relationship would bedemonstra-

ble between the enzymatic activities and the

toxicity of Mamushi venom, especially in

connection with itshemorrhagic andnecrotic

activities.

In this paper, thechromatographic sepa-

rationofthe three proteinases from Mamushi

XII.Murata, Y. , Satake, M., andSuzuki,T. ,

/ .

Biochm.,

53, 431(1963)

* A part of thu paper wai reported orally at

the

13th

Japan Pharmaceutical Assembly (1960)

Th eabbreviations 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;

A TEE,

or-acetyl-L-tyrosine ethyl ester.

venom

is

described.

The

differences

in

their

stabilityand the inhibitory effects of various

reagents, especially of cysteine, will be pre-

sented. Allofthese proteinases were inhibited

by EDTA.

The

proteinases

in

Mamushi

venom seem

to be of a

different type from

the known digestive enzymes such astrypsin

[EC 3.4.4.4].

EXPERIMENTAL

Snake VenomandAntiurumThe venom

of

Japa-

n a e Agkistrodon halys biomhoffii (Mamushi), frozen

immediately after collection, was lyophilized and

uied. Mamushi antiserumwas a productofTakeda

Pharmaceutical Industries, Ltd., prepared from horse

i:mm immunized only with Mamushi venom. Its

potency was 300I.V.LD

U

units/mL

SubstraUs

Casein

according to Hammarstenwas

a product

of E.

Merck

tc Co.

Crystalline murami-

due [EC3.2.1.17] wasprepared by the method of

Aldert on it al.(5). Examinationofits N-terminal

amino acid by the dinitrophenylation method of

S i n g e 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 Peterson

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 Reagents

Metal 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 contentin the

438

atPennStateUniversity(PaternoLib)onJanuary22,2013

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Snake Venom Proteinases. X II I

4 9

cbromatographic fractions was estimated by its UV -

absorption at

280

rapt,based on the absorbancy of

1 mg ./m l. 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**.

T O avoid th e 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

th e

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 d etected in the effluent.

Th e 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 follow s: Th e elution was started

with 330 ml. of 0.005 Mbuffer in the mixing chamber

and 0.1M 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 9 11ml. per hour and

6.5 ml. fractions were c ollected. All procedures were

carried out in the cold room at 4C.

For the fractionation of 1g. of venom , the

column size was enlarged to 2 .2x 38 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 Column Chromatographic 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 ti e 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). Th e other

two peaks of BAEE hydrolyzing activity were

distributed in different fractions from those

contain ing 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< U i / -+ toH / -

60 90 120

TUBE NUMBER

Fio.

la. Chromatographic separation of pro-

teinases on DEAE-cellulose column starting from

200 mg. of M amushi venom.

200 mg. of Mamushi venom was applied on

a DEAE-cellulose column (1.3X 16.5cm .), and

elution was carried out with concen tration

gradient of Na acetate buffer, pH 7.0, from

0.005 M to 0.5 Id as described in experim ental.

Flow rate, 9-11 ml./hour; collection of effluent,

6.5 ml ./tu be. Proteinase activity was illustrated

by AEgao/15 minu tes/0.5 ml. fraction.



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44

M. SATAKE, Y. MURATA and T. SUZUKI

6 0

e o 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:

an d in Fig. lb, Nos.1015as 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,andwere 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

39 0

BA A

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 a

The

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

i

4 0

ISO0 I fO

TUBE NUMBER

Fio. 2a. Rechromatography of proteinase

m

on CM-celluloie.

28.3 mg. of proteinase a recovered in the

fint DEA E-cellulose chromatography wa i applied

on a CM-cellulose column (1.3 x21.O cm.) equi-

librated with 0.005 M Na acetate buffer (pH

5.8). Gradient elution w u 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 12ml./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

chrom atograph y. Proteinase a was found to

be accompanied by BAEE hydrolyzing activity

during the chromatographic fractionation.

Richrom atographiss 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 an y

case,the activities of proteinases b and c emerg-

0 .9

to0 .5

0 . 9 -

|

0.6 J

g

30

ISO

z

o E

SO O ISO

TUBE NUMBER

Fio.

2b. Rechromatography of proteinase a

on phospho-celluloie.

173mg. of proteinase a recovered in the

first DEAE-cellulose chromatography was applied

on a phospho-cellulose column (1.5 x22 .5 cm.)

equilibrated with 0.005 M Na acetate buffer,

pH 6.0. Gradient elution w as carried out with

700ml. 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 1015ml./hour , and

each fraction contained 10ml. Proteinase activity

was illustrated by A E ^/ IS minutes/0.5 ml.

fraction.

ed in a single peak at approxima tely 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 (10mm. in height),

an intermediate 0.8% agar layer

(30mm.),

and the proteinase solution in the top layer

(25

mm.).

The appearanc e of the precipitation

lines in the intermediate layer by the reac-

tions between the diffused reactan ts was



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O 2 4 8 8 1

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 in and

Murarrd-

dase by Protsinases a,b,andc-.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

4 8 8 1

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

250mL of 0.1 M acetate (pH 7.0) set in the

mixing chamber, and 0.5M 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 4.0mL of enzyme solu-

tion containing one of the following proteinases;

810/tg. of proteinase a, 690pg. of proteinase b,

or 790ftg. of proteinase c. At time intervals

ihown in the figure, 1.0 ml. aliquot was taken

out and added into 1.0ml. 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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44

was in cubated for 12 hours with casein and

the increases in TCA-soluble materials were

determine d at the intervals shown in Fig. 5.

The extent of hydrolysis of casein by pro-

teinase b was apparently twice as great as

those obtaine d 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 figur e, 0.2 ml. aliquot was

taken out and added into 1.0 ml. of 0.44 M TCA.

After standing for 30 min utes, the mixture was

filtered and 0.2 ml. of the filtrat e was taken for

measurement of acid-soluble material by Folin

reaction. Muram idase 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 a t 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 p H region, showing different

pH op tim a; 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 term s of r elative activity to the maximum

activity . Th e substrate solutions were made as

follow s: Casein was dissolved at 8% in 0.4 M

Tris-H Cl buffer of pH varying from 7.2 to 9.0,

and h eated 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 NaO H before dilution. All

casein solutions were checked for their pH before

use.

: proteinase a, O : proteinase b,

X : proteinase c.

Stability

Differe nces in heat stability

am ong proteinases a, b and c were revealed

by heat ing for 10 minutes in the range of

4080C as shown in F ig. 8. The results

indicated that proteinase c is the least stable

of the three. Th e 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

und er 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. MUR ATA and T. SUZUKI

IO O

40 DO 60 70

TEMPtR TURE

C O

SO

Fio. 8. Heat stability of venom proteicasei.

0.5 ml. of enzyme solution wa s heated at

indicated temperature

for 10

minutes

in a

water

bath

an d

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, 0.79rag./ml.

; proteinase

c, 0.73

mg./ml.

T he

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

stimulato ry. All other divalent metal ions

acted as inhibitors. Cd

++

, particularly, had

a strong inhibitory effect (Table II).

Effects of O tfur Ag ntsThe proteinase

activities were strongly inhibited by E DT A,

in agree men t with the results of others

(9

IT).

Proteinase c was inhibited completely by

2 x10 *

M

EDTA . PCM B, a sulfhydryl gro up

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 Tab le IV, revealed distinct

differences amo ng thre e proteinases. Prote in-

ase c was inactivated completely by treatmen t

for 30 minutes with 4x10 ' M cysteine.

Efftct of Antistnm

It has been pointed

out in the previous study that Mamushi

venom antiserum contains several anti-en-

TABLE I I

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, lm l. of 1% casein in 0.1 M Trij-HQ

buffer (pH 8.5) was added an d incubated for 20

minutes at 17*0. Then 3 ml. of 5% TCA was

added, and the mixture w as centrifuged after

standing for 1 hour. Th e TCA-soluble product

wa s determined by measuring th e absorbancy at

280 m/L Th e values were expressed in terms of

th e relative percentage of activity to the control

system (no addition of metal salt). Th e 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,

M n ( 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 Mam ushi

venom. Th e effects of antiserum on the thre e

proteinases are shown in Table V. It was

found that inhib ition by antiserum was

observed with each of the proteinases. Th e

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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5

TABUS

i n

Efftcls qf Various Inhibitors on Vtnom ProUmau Actwtius

Reaction mixtures contained 0.5mL 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 was2 X10-*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

TA BLE IV

Efftct of Cjsttvu on

Vtnom

HoUaau Attimtut

0.3 ml. of enryme solution was pretreated with 0.2 ml. of lx l0 ~*

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.

M aeno

et al. 9).

and Ohsaka

10)

have

reported independently the presence of two

or more proteinases in Trinunsurusvenom.

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 H a m b e r g

tt al. IS) have demonstrated a difference in

heat stability between the activities toward



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446


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 studiesare now in progresson

the specificities of these proteinases using B

chain

of

insulin

and

glucagon. While

we

were employed

in

this specificity studies,

P f l e i d e r e r

it al. {II

published the separa-

tion of three proteinases from

Crotalus atrox

venom- Interesting ly, 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

Mam ushi venom will

be presented

in the

following paper.

SUMMARY

The ve nom of Ag/nstrodm halyt blomhoffti

(M amushi) 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 M g

++

, 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 substra te specifici-

ties from each other. Du ring

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...

J. Pharm. Soc. Japan (in Japanese), 80, 1040

(1960)

2) Boman, H. G., and Kaletta, U., Biochim. it

Biophys. Acta,24, 619 (195 7); BjOrk, W ., and

Boman, H. G., Biochim. it Biophys. Acta, 34,

503 (1959)

3) Alderton, G., Ward, H., and Fevold, H. L.,

/ . Biol.Chtm., 157, 43 (1945)

4) Sanger, F., Biochtm. J., 39, 507 (1945)

( 5 ) Peterson, D. A ., and Sober, H. A., / . Am .

Chtm. Soc.,78, 751 (1956)

(6 ) Murata, Y., Satake, M ., and Suzuki, T., / .

Biochtm. (Tokyo), 53, 431 (1963)

(7 ) Bowen, H. E., / . Immunol., 68, 429 (1952)

(tf) Iizuka, K., Murata, Y., and Satake, M ., / .

Pharm. Soc. Japan(in Japa nese), 80, 1035 (1960)

(9) Maeno, H., Miuuhashi, S., and Sato, R.,

Symposia on Enzymt Chtmiitry(in Japa nese), 12,

95 (1960)

JO ) Obsaka, A., Japantsi J. Mid. Sd and

Biol.,

13,

33 (1960)

11) Pfleiderer, G., and Sumyk, G., Biochim. it

Biophys. Acta, 51, 482 (1961)

12) Kaiser, E., and Michl, H., Di e Biochemie

der Tierischen Gifte, Fran* Deuticke, Vienna

(1958) p. 196

13) H amberg , TJ., and R ocba e Silva, M ., Arch,

inttrn.

pharmatodynamit,

110, 222 (1957)



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Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963