chromatographic sep of proteinases from agkistrodon-jbiochem_satake m 1963
TRANSCRIPT
-
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
1/10
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
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
2/10
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.
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
3/10
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
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
4/10
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
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
5/10
M. SATAKE, Y. MURATA and T. SUZUKI
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.
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
6/10
Snake Venom Proteinases. XIII
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.
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
7/10
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).
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
8/10
Snake Venom Proteinases. XIII
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
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
9/10
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 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.,
],
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/ -
8/12/2019 Chromatographic Sep of Proteinases From Agkistrodon-JBiochem_Satake M 1963
10/10
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)
atPennStateUniversity(PaternoLib)onJanuary22,2013
http://jb.oxfordjournals.org/
Downloaded
from
http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/http://jb.oxfordjournals.org/