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Comparative study of Agkistrodon acutus venoms from china and taiwan
Camp.
Biochem.
0306-4492/84$3.00+ 0.00 6; 1984Pergamon Press Ltd
Vol. 79C. No. 1, pp. 51-57, 1984
Phpsiol.
Printed in Great Britain
COMPARATIVE VENOMS
STUDY FROM
OF AGKISTRODON CHINA
AND
ACUTUS
TAIWAN
YUMIKO KOMORI, TOSHIAKI NIKAI and HISAYOSHI SUGIHARA Department of Microbiology, Faculty of Pharmacy, Meijo Umversity, Tenpaku-Ku, Nagoya, Japan (Received 24 January 1984)
Abstract-l. Three hemorrhagic toxins (AC,-. AC?- and AC,-proteinases) were isolated from the lyophilized venom of Agkistrodon acutus from China using gel filtration on a Sephadex G-75 column, followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, DEAE-Sephacel and DEAE-cellulose. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. 3. Three hemorrhagic toxins possessed both lethal and proteolytic activities. These activities were inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol (p-aminoethyl)N,N,N’,N’tetraacetic acid (EGTA), o-phenanthroline or cysteine, but not by soybean trypsin inhibitor (SBTI), p-chloromercuribenzoate (PCMB) or diisopropyl fluorophosphate (DFP). 4. Molecular weight of hemorrhagic toxins (AC,-, Acz- and AC,-proteinases) were determined to be 24,500, 25,000 and 57,000, respectively. It was found that hemorrhagic toxins have considerable similarity to hemorrhagic toxins isolated by Nikai et al. (1977) and Sugihara et al. (1978, 1979), but AC?- and AC,-proteinases from China and Taiwan are electrophoretically unrelated.
0.5 M NaCl in a total volume of 600 ml (Fig. 2B). For the final step to obtain Ac,-proteinase, the fraction B-2 was against 0.01 M Tris-HCI buffer containing dialysed 0.01 M NaCl (pH 7.2), and applied to a column of DEAESephacel (1.5 x 45 cm) equilibrated with the same buffer. The column was developed with a linear gradient from 0.01 to 0.04 M NaCl in a total volume of 600 ml (Fig. 2C). To obtain AC,-proteinase, the fraction B-l was dialysed against 0.01 M Tris-HCl buffer containing 0.01 M NaCl (pH 7.2), and applied to a column of DEAE-cellulose (1.5 x 45 cm) equilibrated with the same buffer. The column was developed with a linear gradient from 0.01 to 0.3 M NaCl in a total volume of 500ml (Fig. 2D). On the other hand, AC,-proteinase was isolated as follows. The fractions A-l and A-2 were applied to a column of DEAE-Sephadex A-50
INTRODUCTION Agkistrodon acutus (family: Crotalidae) is found in mainland China and Taiwan, but not in the New World, Europe and Africa (Fig. 1). The characteristic local symptoms produced by the bite of Agkistrodon acutus are hemorrhage, necrosis and muscular degeneration. The isolation of hemorrhagic toxins from the venom of Agkistrodon acutus from Taiwan has been reported (Nikai et al., 1977; Sugihara et al., 1978, 1979). In the present paper, we have investigated the isolation and some properties of the three hemorrhagic toxins, AC,-, AC,- and AC,-proteinases from China and differences between hemorrhagic toxins of Agkistrodon ucutus from China and Taiwan. MATERIALS AND METHODS Materials. Lyophilized crude venom of Agkistrodon acutus from China and Taiwan were purchased from the Japan Snake Institute, Gunma. Antivenines (China and Taiwan) were the products of the Institute of Biologics, Shanghai and Taiwan Serum Vaccine Laboratory, Nan-Kang Taipei, respectively. Sephadex G-75, DEAE-Sephadex A-50, DEAESephacel, ampholytes over the pH range 3.5-10 and molecular weight protein standards were obtained from Pharmacia Fine Chemicals. DEAE-cellulose was purchased from Whatman Biochemicals, Ltd. Casein (Hammarsten) was obtained from Merck. Other chemicals used were analytical grade from commercial sources. Isolafion. All fractionation steps were performed at 4°C. Two grams of crude venom was dissolved in 8.0ml of 0.04 M Tri-HCl buffer containing 0.01 M NaCl @H 7.2), and centrifuged at 7,000 rpm for 30 min. The supernatant was applied to a gel-filtration column of Sephadex G-75 (5.0 x 90cm) equilibrated with the same buffer (Fig. 2A). The fraction A-4 was applied to a column of DEAESephadex A-50 (1.9 x 45 cm) equilibrated with 0.04 M Tris-HCl buffer containing 0.01 M NaCl @H 7.2). The column was developed with a linear gradient from 0.01 to
Fig. 1. Geographical distribution of Agkistrodon acutus. (1) Hupii; (2) Anhui; (3) Ssuchuan; (4) Kueichou; (5) Hunan; (6) Chianghsi; (7) ChQhiang; (8) Kuanghsi; (9) Kuangtung; (10) Fuchien; (11) Taiwan. 51
52
YUMIKOKOMORIet al.
Tube Nuder
06’
lubr
E:,wnhm
Fig. 2. Purification scheme of AC,-, AC,- and AC,-proteinases from the venom of Agkistrodon acutus from China. The isolation procedure is described in Materials and Methods. e--O, protein fractions detected by absorbance at 280 nm; O----O, proteolytic activity; ----, conductivity of the eluents. (A) First-step purification using a Sephadex G-75 column. (B) Second-step purification to obtain AC,- and AC,-proteinases using a DEAE-Sephadex A-50 column. (C) Final-step for Ac,-proteinase using a DEAE-Sephacel column. (D) Final-step for AC,-proteinase using a DEAE-cellulose column. (E) Secondstep to obtain AC,-proteinase using a DEAE-Sephadex A-50 column. A......A, Activity of arginine ester hydrolase (China); n-----A, activity of arginine ester hydrolase (Taiwan). (F) and (G) Third and final-steps for AC,-proteinase using a DEAE-Sephacel column.
Agkistrodon acutus venoms
53
“Cys/Z content was determined as S-carboxymethlcysteine ‘Tryptophan content was determined by U.V. absorption.
the geis into a mixture of 7.5fi; acetic acid and 5% methanol in H,O. Isoefectricpoint, The isoelectric points of purified enzymes were estimated by using an isoelectric focusing column of volume of 800 ml (Fig. 2E). The last fraction of Fig. 2E was dialysed against 0.01 M Tris-HCI bufler containing LKB Instruments. The ampholyte concentration was 2% 0.01 M NaCl (pH 7.21, and applied to a column of DEAE(w/v), and the pH ranged from 3 to IO. At the end of the experiments, the column was drained, and 2.0 ml fractions Sephacef (I.5 x 4Scm) ~~~ilibra~~ with the same buffer. The column was developed with a linear gradient from 0.1 were collected. Absorption at 280 nm was monitored by a to 0.6 M NaCl in a total volume of 600 ml (Fig. 2F). For the spectrophotometer. The pH of each 2 ml fraction was also final step to obtain AC,-proteinase, the fraction F-3 was measured. purified by the same procedure on DEAE-Sephncel column Molecular weight. The molecular weight was estimated by SDS-potyacrylamide gel electrophoresis using 8% poiyrechromatography (Fig. 2G). ~e~~o~~~ugic a&&y. Hemorrhagic activity was zssayed acryfamide gel. Protein standards used were phosphorylase by the method of Kondo ef ai. (1960). The sample was b (94~~0~, bovine serum albumin (67,000), ovalbumin diluted three-fold, and 0.1 ml of each dilution was injected (43,000), carbonic anhydrase (30,000), soybean trypsin ini.c. into the depilated skin of the back of albino rabbits. hibitor (20,100) and a-lactalbumin (14,400). After 24 hr, the depilated skin was removed and the Carbohydrate. The neutral sugar content was analysed by diameter of each hemorrhagic spot was measured. The the method of Morris (1948). activity was expressed as Erg of protein~MH~, MHD E&cts of‘ inhibitors. Effects of inhibitors on the hemorr~min~mum hemorrhagic dose) was defined as the a~eunt of hagic, Iethal and proteotytic activities were assayed as protein that produced a hemorrhagic lesion of IOmm in follows: each inhibitor was incubated with Ac,-proteinase {for 15 min), Ac,-proteinase (for 30 min) and Ac,-proteinase diameter. (for 5 min) at 3?C. After these preincubations each sample Lethal octiuity. Lethal activity was assayed by following procedure. Mice (16-17 g) were injected i.v. with 0.21~11 was assayed for hemorrhagic. lethal and proteolytic activities respectively. venom solutions which were diluted i.J-fold (eight mice per Amino acid co~~os~tio~s. Amino acid compositions were dose). Mice were observed up to 4 days after injection and determined with a Hitachi Model 835 automatic amino acid the LB,, values were calculated by the method of Litchfield analyser. Hydrolysis of samples was done with constant and Wilcoxon (1949). boiling HCl at 110°C for 24, 48 and 72 hr. Proteolytic activity. Proteolytic activity was assayed by the method of Murata ef al. (1963). Casein. 1.05 g in 50 ml of 0.4 M fris-HC1 bufIer (pfi 8.5), was dissolved by heating for 15 min in a boiling water bath. For dete~jnat~on of casein digestion, 0.5 ml of the enzyme solution and the 2% Isolation casein solution were incubated for 15 min at 37°C. The Purification of A+proteinase was achieved in reaction was stopped by adding I ml of trichloroacetic acid three steps (Fig. 2A, 2B and 2C). The final fraction (TCA) and left to stand for 30 min, after which the mixture (Fig. 2C) possesses hemorrhagic, lethal and prowas filtered in Whatman No. 2 filter paper. Two and a half rn~~~~l~t~s of 0.4 M Na#IQ and 0.5 ml of Folin’s reagent teolytic activities. Each activity was 1.22 j6g (MHD), (diluted to l/3 original strength) were added to 0.5mi of 5.3 (357.0) fig/g mice (LD,,) and 0.315 units/mg filtrate and the color that developed was read at 660 nm. The respectively (Table 2). In the second purification step, enzyme activity was obtained by means of the following two fractions show proteolytic activity (Fig. 2B). equation: units/mg = absorbance change per min x 2/mg. AC,-Proteinase was obtained from the fraction of Ho?nogeneQ. The disc gel ebctrophoresis was carried out peak 1 followed by chromatography on DEAEon ~Iya~~iam~de get, using the Tris-glycine system lethal (pH 8.31, at 4°C for 4 hr by applying 1.5 mA/tube. The gels cellulose (Fig. 2D). It possesses hemorrhagic, and proteolytic activities and each activity was were stained with Coomassie brilliant blue R-250 (Sigma), and excess dye was removed by allowing it to diffuse out of 1.21 pg (MIX)), 10.3 (9.8-10.8) pg/g mice (LD,,) and f I.9 x 45 cm) equilibrate& with 0.04 M Tris-HCI b&er containing 0.01 M NaC‘I (pH 7.2’). The column was developed with a linear gradient from O-01 to 0.6 M NaCl in a total
54
YUMIKO KOMORI el Table 2. Comparison
of AC,-, AC,- and AC,-proteinases
from Agkistrodon
AC,-Proteinase Property
and
Taiwan
activity
Taiwan
(Taiwan
and China)
AC3-PWJteiIidW
Chl"a
weight
~CUIUS venoms -
Ac2-Prote3nase
China
Molecular
ai.
TaiWa"
_..-____._~___-.___
Chl"d ~~~~~~~.._
24500
24500
25000
zsoofl
57000
57000
4.7
4.7
4.9
5.1
4.1
5.0
Disc electrophorerir (Rf) Carbohydrate content
0.69
0.69
0.57
0.51
0.49
0.46
no
"0
"a
no
1.0
1.c"
Optimum
8.5
8.5
8.5
8.5
8.5
8.5
45oc
45T
37iC
37T
28T
28OC
5-8
5-8
6-8
6.)
6-10
6-10
pmteolytlc actxvity (unit/mg)
0.544
0.315
0.173
0.189
0.253
0.315
tiemrrhagic act1uity (tI.H.D..ug)
0.22
1.22
0.43
1.21
0.95
1.53
Isoelectric
Heat
point
pH
stability
pH stability
Ouchterlony double
diffusion
Lethdl activity (LD5O*ug/g) Inhibitor
(3X1) EOTA.EGTA, o-Phenanthroline. cyrteine
(3.Z.0, EOTA,EGTA, o-Pbenanthrollne. CySteille
10.3 (9.8-10.8)
,6.:::.2, EDTA.EGTA, o-Pbenanthroline. cysttine
EDTA.EGTA, o-Phcnanthroline. cysteine
7.2 16.6-7.9) EDTA.LGTA, o-Phenanthraline. Cysteine
11.3 (10.5-12.2) EDTA,EGTA, o-Phenanthroline, Cystcine
0.189 units/mg severally (Table 2). Purification of Ac,-proteinase was achieved in four steps (Figs 2A, 2E, 2F and 2G). The final fraction (Fig. 2G) possesses hemorrhagic, lethal and proteolytic activities, and each activity of them was 1.53 pg (MHD), 11.3 (10.5-12.2) pg/g mice (LD,,) and 0.315 units/mg respectively (Table 2). Each purification step shows the same pattern as the pattern of the Agkistrodon acutus venom from Taiwan.
the phylogenetic kinship of snakes, each of the China and Taiwan proteinases fused with the antivenines of China and Taiwan (Fig. 4). Judging from precipitin lines, Ac,-proteinases of China and Taiwan were identical. Similarly about AC,- and Ac,-proteinases, the precipitin lines of China and Taiwan were fused. These results show that AC,- and AC,-proteinases have a common antigen respectively between China and Taiwan, whereas they have different isoelectric points.
Each proteinase shows only one band by disc electrophoresis (Fig. 3). The mobilities of AC,-, AC*and AC,-proteinases were compared between China and Taiwan. As shown in Fig. 3A, Ac,-proteinases of China and Taiwan were homogeneous. On the other hand, AC,- and AC,-proteinases were different between China and Taiwan in their mobilities (Figs 3B and 3C). These facts were supported by the results of isoelectric focusings (Table 2). The isoelectric points of Ac,-proteinases were both 4.7, and it shows that China and Taiwan are homogeneous. However, the isoelectric point of AC,-proteinase from China was shown to be 5.1 compared with Taiwan which was shown to be 4.9. Similarly, AC,-proteinase from China was also a little more basic than that from Taiwan, each isoelectric point was 5.0 and 4.7, respectively.
Effects of inhibitors
Amino acid compositions
The amino acid compositions of each proteinase are shown in Table 1. The total residues per mole of Ac,-proteinases were given 227 and 228, AC,-proteinases were given 229 and 230 and AC,-proteinases were given 507 and 508. The results were fitted in the molecular weight of each proteinase (24,500, 25,000 and 57,000; shown in Table 2) determined by the SDS-polyacryiamide gel method. The amino acid composition of each proteinase from China was similar to the proteinases from Taiwan, except for a little difference about serine residues (AC,-proteinase), and glycine. cysteine, valine and isoleucine (Act-proteinase).
Effects of inhibitors on all preteinases were investigated, using EDTA, EGTA, o-phenanthroline, cysteine, SBTI, DFP and PCMB as inhibitor. All proteinases from both China and Taiwan were inhibited in their hemorrhage, lethal and proteolytic activities by EDTA, EGTA, o-phenanthroline and cysteine. These facts indicate that each proteinase has some metals on its active site, and some disulfide-bonds have an important role to keep the structures of these proteinases intact. Friedrich and Tu (1971) reported that Agkistrodon acutus venom from Taiwan includes Ca, Zn and Mg ions, and Nikai ef al. (1982) determined the zinc content of AC,proteinase from Taiwan. According to the results of atomic absorption, AC,-, AC,- and AC,-proteinases contained both Zn and Ca ions. As hemorrhagic, lethal and proteolytic effects were inhibited simultaneously, it is indicated that these effects caused by the same active sites. Other chemicul charac~er~~a~~on
Purified AC,- and AC,-proteinases contain no carbohydrate, whereas the carbohydrate content of AC,-proteinases from China and Taiwan were both found to be 1% (w/w) of protein (Table 2). The activities of the three proteinases decrease at temperatures higher than 45°C 37°C and 28°C respectively. All enzymes were stable at a medium pH range, with their optimum activities at pH 8.5 (Table 2). DISCUSSION
Immunodijjiision
As immunology is a useful technique to investigate
The comparison
of Agkistrodon
acutus
venoms
Agkistrodon
acutus venoms
of AC,-, AC,- and Aqproteinases from China and Taiwan. (A) Fig. 3. Disc-gel electrophoresis [l] Taiwan, [2] China and Taiwan, [3] China. (B) Aqproteinase: [I] Taiwan, [2] China AC,-r xoteinase; and Taiwan, [3] China. (C) AC,-proteinase: [I] Taiwan, [2] China and Taiwan, [3] China.
55
56
YUMIKO KOMOKI etal.
Fig. 4. Immunodiffusion of anti-ilgkistrou’on acutus venoms from China and Taiwan. (C) Hemorrhagic proteinase from China. (T) hemorrhagic proteinase from Taiwan. Immunodiffusion of anti-A. acuius venom (Taiwan) against [A] AC,-protcinase, [C] A+proteinase, and [E] Aqproteinase. Immunodiffusion of anti-d. ac~~us venom (China) against [B] Aqproteinase, [D] Ac,-proteinase, and [F] Aqproteinase.
Agkistrodon acutus venoms from China and Taiwan is valuable because Taiwan was separated from mainland China about a million years ago. Our results indicate that AC,-proteinases from China and Taiwan should be considered to be identical by using disc-gel electrophoresis, isoelectric focusing, immunology and other chemical characterizations. Besides, AC,- and AC,-proteinases are little different between China and Taiwan according to the results of disc-gel electrophoresis and isoelectric focusing, however they are found to have no immunological differences. These facts signify that AC,and AC,-proteinases from China and Taiwan have a common antigenicity. The differences of mobilities of disc-gel electrophoresis and isoelectric points may depend on the changes of some amino acid residues, which happened at another site than that which shows antigenicity. Tu et al. (1980) reported about Trimeresurus jfavoviridis that the venom from Ie Island shows a different pattern by isotachophoresis from the other Trimeresurus javoviridis venoms, in spite of having no immunological difference. In both and Trimeresurus cases Agkistrodon acutus jfavoviridis, some differences are produced through geographical separation, because the sea is the most effective barrier for terrestrial venomous snakes. The physiological effects of Agkistrodon acutus venoms from China and Taiwan are similar, for example, AC,-proteinases attacks kidney, AC,-proteinases attacks intestine and AC,-proteinases attacks lungs (Homma et al., 1980). The results mentioned above indicate that the venoms from China and Taiwan are almost similar, but in detail they are different.
REFERENCES
Friedrich C. and Tu A. T. (1971) Role of metals in snake
57
venoms for hemorrhagic, esterase and proteolytic activities Biochem. Pharmac. 20, 1549-l 5561 Homma M.. Kubota F.. Nikai T. and Sueihara H. (1980) Pathologic changes produced by loo-pacer snake venom and its purified proteinases; with special reference to hemorrhagic lesion. Kitakanto Iguku 30, 485494. Kondo H., Kondo S., Ikezawa H., Murata R. and Oshaka A. (1960) Studies on the quantitative method for the determination of hemorrhagic activity of Habu snake venom. Jap. J. med. Sri. Biol. 13, 43-51. Litchfield J. R. and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96, 99-113. Morris 0. L. (1948) Quantitative determination of carbohydrates with Dreywoods Anthrone reagent. Science 107, 254-255. Murata Y., Satake M. and Suzuki T. (1963) Studies on snake venom. Distribution of proteinase activities among Japanese and Formosan snake venoms. J. Biochem. 53,
431437. Nikai T., Sugihara H. and Tanaka T. (1977) Enzymochemical studies on snake Venoms. II. Purification of lethal protein Ac,-proteinase in the venom of Agkistrodon acutus. Yakugaku Zasshi 97, 507-514. Nikai T., Ishizaki H., Tu A. T. and Sugihara H. (1982) Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom. Comp. Biochem. Physiol.
72C, 103-106. Sugihara H., Nikai T., Kawaguchi T. and Tanaka T. (1978) Enzymochemical studies on snake venoms. IV. Purification of lethal protein AC,-proteinase in the venom of Agkbtrodon acutus. Yakugaku Zasshi 98, 1523-1529. Sugihara H., Nikai T., Umeda H. and Tanaka T. (1979) Enzymochemical studies on snake venoms. V. Purification of lethal protein AC,-proteinase in the venom of Agklrtrodon acutus. Yakugaku Zasshi 99, 1161-l 167. Tu A. T., Stermitz J. and Ishizaki H. (1980) Comparative study of pit viper venoms of Genera Trimeresurus from Asia and Bothrops from America: An immunological and isotachophoretic study. Comp. Biochem. Physiol. 66B, 249-254.
Biochem.
0306-4492/84$3.00+ 0.00 6; 1984Pergamon Press Ltd
Vol. 79C. No. 1, pp. 51-57, 1984
Phpsiol.
Printed in Great Britain
COMPARATIVE VENOMS
STUDY FROM
OF AGKISTRODON CHINA
AND
ACUTUS
TAIWAN
YUMIKO KOMORI, TOSHIAKI NIKAI and HISAYOSHI SUGIHARA Department of Microbiology, Faculty of Pharmacy, Meijo Umversity, Tenpaku-Ku, Nagoya, Japan (Received 24 January 1984)
Abstract-l. Three hemorrhagic toxins (AC,-. AC?- and AC,-proteinases) were isolated from the lyophilized venom of Agkistrodon acutus from China using gel filtration on a Sephadex G-75 column, followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, DEAE-Sephacel and DEAE-cellulose. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. 3. Three hemorrhagic toxins possessed both lethal and proteolytic activities. These activities were inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol (p-aminoethyl)N,N,N’,N’tetraacetic acid (EGTA), o-phenanthroline or cysteine, but not by soybean trypsin inhibitor (SBTI), p-chloromercuribenzoate (PCMB) or diisopropyl fluorophosphate (DFP). 4. Molecular weight of hemorrhagic toxins (AC,-, Acz- and AC,-proteinases) were determined to be 24,500, 25,000 and 57,000, respectively. It was found that hemorrhagic toxins have considerable similarity to hemorrhagic toxins isolated by Nikai et al. (1977) and Sugihara et al. (1978, 1979), but AC?- and AC,-proteinases from China and Taiwan are electrophoretically unrelated.
0.5 M NaCl in a total volume of 600 ml (Fig. 2B). For the final step to obtain Ac,-proteinase, the fraction B-2 was against 0.01 M Tris-HCI buffer containing dialysed 0.01 M NaCl (pH 7.2), and applied to a column of DEAESephacel (1.5 x 45 cm) equilibrated with the same buffer. The column was developed with a linear gradient from 0.01 to 0.04 M NaCl in a total volume of 600 ml (Fig. 2C). To obtain AC,-proteinase, the fraction B-l was dialysed against 0.01 M Tris-HCl buffer containing 0.01 M NaCl (pH 7.2), and applied to a column of DEAE-cellulose (1.5 x 45 cm) equilibrated with the same buffer. The column was developed with a linear gradient from 0.01 to 0.3 M NaCl in a total volume of 500ml (Fig. 2D). On the other hand, AC,-proteinase was isolated as follows. The fractions A-l and A-2 were applied to a column of DEAE-Sephadex A-50
INTRODUCTION Agkistrodon acutus (family: Crotalidae) is found in mainland China and Taiwan, but not in the New World, Europe and Africa (Fig. 1). The characteristic local symptoms produced by the bite of Agkistrodon acutus are hemorrhage, necrosis and muscular degeneration. The isolation of hemorrhagic toxins from the venom of Agkistrodon acutus from Taiwan has been reported (Nikai et al., 1977; Sugihara et al., 1978, 1979). In the present paper, we have investigated the isolation and some properties of the three hemorrhagic toxins, AC,-, AC,- and AC,-proteinases from China and differences between hemorrhagic toxins of Agkistrodon ucutus from China and Taiwan. MATERIALS AND METHODS Materials. Lyophilized crude venom of Agkistrodon acutus from China and Taiwan were purchased from the Japan Snake Institute, Gunma. Antivenines (China and Taiwan) were the products of the Institute of Biologics, Shanghai and Taiwan Serum Vaccine Laboratory, Nan-Kang Taipei, respectively. Sephadex G-75, DEAE-Sephadex A-50, DEAESephacel, ampholytes over the pH range 3.5-10 and molecular weight protein standards were obtained from Pharmacia Fine Chemicals. DEAE-cellulose was purchased from Whatman Biochemicals, Ltd. Casein (Hammarsten) was obtained from Merck. Other chemicals used were analytical grade from commercial sources. Isolafion. All fractionation steps were performed at 4°C. Two grams of crude venom was dissolved in 8.0ml of 0.04 M Tri-HCl buffer containing 0.01 M NaCl @H 7.2), and centrifuged at 7,000 rpm for 30 min. The supernatant was applied to a gel-filtration column of Sephadex G-75 (5.0 x 90cm) equilibrated with the same buffer (Fig. 2A). The fraction A-4 was applied to a column of DEAESephadex A-50 (1.9 x 45 cm) equilibrated with 0.04 M Tris-HCl buffer containing 0.01 M NaCl @H 7.2). The column was developed with a linear gradient from 0.01 to
Fig. 1. Geographical distribution of Agkistrodon acutus. (1) Hupii; (2) Anhui; (3) Ssuchuan; (4) Kueichou; (5) Hunan; (6) Chianghsi; (7) ChQhiang; (8) Kuanghsi; (9) Kuangtung; (10) Fuchien; (11) Taiwan. 51
52
YUMIKOKOMORIet al.
Tube Nuder
06’
lubr
E:,wnhm
Fig. 2. Purification scheme of AC,-, AC,- and AC,-proteinases from the venom of Agkistrodon acutus from China. The isolation procedure is described in Materials and Methods. e--O, protein fractions detected by absorbance at 280 nm; O----O, proteolytic activity; ----, conductivity of the eluents. (A) First-step purification using a Sephadex G-75 column. (B) Second-step purification to obtain AC,- and AC,-proteinases using a DEAE-Sephadex A-50 column. (C) Final-step for Ac,-proteinase using a DEAE-Sephacel column. (D) Final-step for AC,-proteinase using a DEAE-cellulose column. (E) Secondstep to obtain AC,-proteinase using a DEAE-Sephadex A-50 column. A......A, Activity of arginine ester hydrolase (China); n-----A, activity of arginine ester hydrolase (Taiwan). (F) and (G) Third and final-steps for AC,-proteinase using a DEAE-Sephacel column.
Agkistrodon acutus venoms
53
“Cys/Z content was determined as S-carboxymethlcysteine ‘Tryptophan content was determined by U.V. absorption.
the geis into a mixture of 7.5fi; acetic acid and 5% methanol in H,O. Isoefectricpoint, The isoelectric points of purified enzymes were estimated by using an isoelectric focusing column of volume of 800 ml (Fig. 2E). The last fraction of Fig. 2E was dialysed against 0.01 M Tris-HCI bufler containing LKB Instruments. The ampholyte concentration was 2% 0.01 M NaCl (pH 7.21, and applied to a column of DEAE(w/v), and the pH ranged from 3 to IO. At the end of the experiments, the column was drained, and 2.0 ml fractions Sephacef (I.5 x 4Scm) ~~~ilibra~~ with the same buffer. The column was developed with a linear gradient from 0.1 were collected. Absorption at 280 nm was monitored by a to 0.6 M NaCl in a total volume of 600 ml (Fig. 2F). For the spectrophotometer. The pH of each 2 ml fraction was also final step to obtain AC,-proteinase, the fraction F-3 was measured. purified by the same procedure on DEAE-Sephncel column Molecular weight. The molecular weight was estimated by SDS-potyacrylamide gel electrophoresis using 8% poiyrechromatography (Fig. 2G). ~e~~o~~~ugic a&&y. Hemorrhagic activity was zssayed acryfamide gel. Protein standards used were phosphorylase by the method of Kondo ef ai. (1960). The sample was b (94~~0~, bovine serum albumin (67,000), ovalbumin diluted three-fold, and 0.1 ml of each dilution was injected (43,000), carbonic anhydrase (30,000), soybean trypsin ini.c. into the depilated skin of the back of albino rabbits. hibitor (20,100) and a-lactalbumin (14,400). After 24 hr, the depilated skin was removed and the Carbohydrate. The neutral sugar content was analysed by diameter of each hemorrhagic spot was measured. The the method of Morris (1948). activity was expressed as Erg of protein~MH~, MHD E&cts of‘ inhibitors. Effects of inhibitors on the hemorr~min~mum hemorrhagic dose) was defined as the a~eunt of hagic, Iethal and proteotytic activities were assayed as protein that produced a hemorrhagic lesion of IOmm in follows: each inhibitor was incubated with Ac,-proteinase {for 15 min), Ac,-proteinase (for 30 min) and Ac,-proteinase diameter. (for 5 min) at 3?C. After these preincubations each sample Lethal octiuity. Lethal activity was assayed by following procedure. Mice (16-17 g) were injected i.v. with 0.21~11 was assayed for hemorrhagic. lethal and proteolytic activities respectively. venom solutions which were diluted i.J-fold (eight mice per Amino acid co~~os~tio~s. Amino acid compositions were dose). Mice were observed up to 4 days after injection and determined with a Hitachi Model 835 automatic amino acid the LB,, values were calculated by the method of Litchfield analyser. Hydrolysis of samples was done with constant and Wilcoxon (1949). boiling HCl at 110°C for 24, 48 and 72 hr. Proteolytic activity. Proteolytic activity was assayed by the method of Murata ef al. (1963). Casein. 1.05 g in 50 ml of 0.4 M fris-HC1 bufIer (pfi 8.5), was dissolved by heating for 15 min in a boiling water bath. For dete~jnat~on of casein digestion, 0.5 ml of the enzyme solution and the 2% Isolation casein solution were incubated for 15 min at 37°C. The Purification of A+proteinase was achieved in reaction was stopped by adding I ml of trichloroacetic acid three steps (Fig. 2A, 2B and 2C). The final fraction (TCA) and left to stand for 30 min, after which the mixture (Fig. 2C) possesses hemorrhagic, lethal and prowas filtered in Whatman No. 2 filter paper. Two and a half rn~~~~l~t~s of 0.4 M Na#IQ and 0.5 ml of Folin’s reagent teolytic activities. Each activity was 1.22 j6g (MHD), (diluted to l/3 original strength) were added to 0.5mi of 5.3 (357.0) fig/g mice (LD,,) and 0.315 units/mg filtrate and the color that developed was read at 660 nm. The respectively (Table 2). In the second purification step, enzyme activity was obtained by means of the following two fractions show proteolytic activity (Fig. 2B). equation: units/mg = absorbance change per min x 2/mg. AC,-Proteinase was obtained from the fraction of Ho?nogeneQ. The disc gel ebctrophoresis was carried out peak 1 followed by chromatography on DEAEon ~Iya~~iam~de get, using the Tris-glycine system lethal (pH 8.31, at 4°C for 4 hr by applying 1.5 mA/tube. The gels cellulose (Fig. 2D). It possesses hemorrhagic, and proteolytic activities and each activity was were stained with Coomassie brilliant blue R-250 (Sigma), and excess dye was removed by allowing it to diffuse out of 1.21 pg (MIX)), 10.3 (9.8-10.8) pg/g mice (LD,,) and f I.9 x 45 cm) equilibrate& with 0.04 M Tris-HCI b&er containing 0.01 M NaC‘I (pH 7.2’). The column was developed with a linear gradient from O-01 to 0.6 M NaCl in a total
54
YUMIKO KOMORI el Table 2. Comparison
of AC,-, AC,- and AC,-proteinases
from Agkistrodon
AC,-Proteinase Property
and
Taiwan
activity
Taiwan
(Taiwan
and China)
AC3-PWJteiIidW
Chl"a
weight
~CUIUS venoms -
Ac2-Prote3nase
China
Molecular
ai.
TaiWa"
_..-____._~___-.___
Chl"d ~~~~~~~.._
24500
24500
25000
zsoofl
57000
57000
4.7
4.7
4.9
5.1
4.1
5.0
Disc electrophorerir (Rf) Carbohydrate content
0.69
0.69
0.57
0.51
0.49
0.46
no
"0
"a
no
1.0
1.c"
Optimum
8.5
8.5
8.5
8.5
8.5
8.5
45oc
45T
37iC
37T
28T
28OC
5-8
5-8
6-8
6.)
6-10
6-10
pmteolytlc actxvity (unit/mg)
0.544
0.315
0.173
0.189
0.253
0.315
tiemrrhagic act1uity (tI.H.D..ug)
0.22
1.22
0.43
1.21
0.95
1.53
Isoelectric
Heat
point
pH
stability
pH stability
Ouchterlony double
diffusion
Lethdl activity (LD5O*ug/g) Inhibitor
(3X1) EOTA.EGTA, o-Phenanthroline. cyrteine
(3.Z.0, EOTA,EGTA, o-Pbenanthrollne. CySteille
10.3 (9.8-10.8)
,6.:::.2, EDTA.EGTA, o-Pbenanthroline. cysttine
EDTA.EGTA, o-Phcnanthroline. cysteine
7.2 16.6-7.9) EDTA.LGTA, o-Phenanthraline. Cysteine
11.3 (10.5-12.2) EDTA,EGTA, o-Phenanthroline, Cystcine
0.189 units/mg severally (Table 2). Purification of Ac,-proteinase was achieved in four steps (Figs 2A, 2E, 2F and 2G). The final fraction (Fig. 2G) possesses hemorrhagic, lethal and proteolytic activities, and each activity of them was 1.53 pg (MHD), 11.3 (10.5-12.2) pg/g mice (LD,,) and 0.315 units/mg respectively (Table 2). Each purification step shows the same pattern as the pattern of the Agkistrodon acutus venom from Taiwan.
the phylogenetic kinship of snakes, each of the China and Taiwan proteinases fused with the antivenines of China and Taiwan (Fig. 4). Judging from precipitin lines, Ac,-proteinases of China and Taiwan were identical. Similarly about AC,- and Ac,-proteinases, the precipitin lines of China and Taiwan were fused. These results show that AC,- and AC,-proteinases have a common antigen respectively between China and Taiwan, whereas they have different isoelectric points.
Each proteinase shows only one band by disc electrophoresis (Fig. 3). The mobilities of AC,-, AC*and AC,-proteinases were compared between China and Taiwan. As shown in Fig. 3A, Ac,-proteinases of China and Taiwan were homogeneous. On the other hand, AC,- and AC,-proteinases were different between China and Taiwan in their mobilities (Figs 3B and 3C). These facts were supported by the results of isoelectric focusings (Table 2). The isoelectric points of Ac,-proteinases were both 4.7, and it shows that China and Taiwan are homogeneous. However, the isoelectric point of AC,-proteinase from China was shown to be 5.1 compared with Taiwan which was shown to be 4.9. Similarly, AC,-proteinase from China was also a little more basic than that from Taiwan, each isoelectric point was 5.0 and 4.7, respectively.
Effects of inhibitors
Amino acid compositions
The amino acid compositions of each proteinase are shown in Table 1. The total residues per mole of Ac,-proteinases were given 227 and 228, AC,-proteinases were given 229 and 230 and AC,-proteinases were given 507 and 508. The results were fitted in the molecular weight of each proteinase (24,500, 25,000 and 57,000; shown in Table 2) determined by the SDS-polyacryiamide gel method. The amino acid composition of each proteinase from China was similar to the proteinases from Taiwan, except for a little difference about serine residues (AC,-proteinase), and glycine. cysteine, valine and isoleucine (Act-proteinase).
Effects of inhibitors on all preteinases were investigated, using EDTA, EGTA, o-phenanthroline, cysteine, SBTI, DFP and PCMB as inhibitor. All proteinases from both China and Taiwan were inhibited in their hemorrhage, lethal and proteolytic activities by EDTA, EGTA, o-phenanthroline and cysteine. These facts indicate that each proteinase has some metals on its active site, and some disulfide-bonds have an important role to keep the structures of these proteinases intact. Friedrich and Tu (1971) reported that Agkistrodon acutus venom from Taiwan includes Ca, Zn and Mg ions, and Nikai ef al. (1982) determined the zinc content of AC,proteinase from Taiwan. According to the results of atomic absorption, AC,-, AC,- and AC,-proteinases contained both Zn and Ca ions. As hemorrhagic, lethal and proteolytic effects were inhibited simultaneously, it is indicated that these effects caused by the same active sites. Other chemicul charac~er~~a~~on
Purified AC,- and AC,-proteinases contain no carbohydrate, whereas the carbohydrate content of AC,-proteinases from China and Taiwan were both found to be 1% (w/w) of protein (Table 2). The activities of the three proteinases decrease at temperatures higher than 45°C 37°C and 28°C respectively. All enzymes were stable at a medium pH range, with their optimum activities at pH 8.5 (Table 2). DISCUSSION
Immunodijjiision
As immunology is a useful technique to investigate
The comparison
of Agkistrodon
acutus
venoms
Agkistrodon
acutus venoms
of AC,-, AC,- and Aqproteinases from China and Taiwan. (A) Fig. 3. Disc-gel electrophoresis [l] Taiwan, [2] China and Taiwan, [3] China. (B) Aqproteinase: [I] Taiwan, [2] China AC,-r xoteinase; and Taiwan, [3] China. (C) AC,-proteinase: [I] Taiwan, [2] China and Taiwan, [3] China.
55
56
YUMIKO KOMOKI etal.
Fig. 4. Immunodiffusion of anti-ilgkistrou’on acutus venoms from China and Taiwan. (C) Hemorrhagic proteinase from China. (T) hemorrhagic proteinase from Taiwan. Immunodiffusion of anti-A. acuius venom (Taiwan) against [A] AC,-protcinase, [C] A+proteinase, and [E] Aqproteinase. Immunodiffusion of anti-d. ac~~us venom (China) against [B] Aqproteinase, [D] Ac,-proteinase, and [F] Aqproteinase.
Agkistrodon acutus venoms from China and Taiwan is valuable because Taiwan was separated from mainland China about a million years ago. Our results indicate that AC,-proteinases from China and Taiwan should be considered to be identical by using disc-gel electrophoresis, isoelectric focusing, immunology and other chemical characterizations. Besides, AC,- and AC,-proteinases are little different between China and Taiwan according to the results of disc-gel electrophoresis and isoelectric focusing, however they are found to have no immunological differences. These facts signify that AC,and AC,-proteinases from China and Taiwan have a common antigenicity. The differences of mobilities of disc-gel electrophoresis and isoelectric points may depend on the changes of some amino acid residues, which happened at another site than that which shows antigenicity. Tu et al. (1980) reported about Trimeresurus jfavoviridis that the venom from Ie Island shows a different pattern by isotachophoresis from the other Trimeresurus javoviridis venoms, in spite of having no immunological difference. In both and Trimeresurus cases Agkistrodon acutus jfavoviridis, some differences are produced through geographical separation, because the sea is the most effective barrier for terrestrial venomous snakes. The physiological effects of Agkistrodon acutus venoms from China and Taiwan are similar, for example, AC,-proteinases attacks kidney, AC,-proteinases attacks intestine and AC,-proteinases attacks lungs (Homma et al., 1980). The results mentioned above indicate that the venoms from China and Taiwan are almost similar, but in detail they are different.
REFERENCES
Friedrich C. and Tu A. T. (1971) Role of metals in snake
57
venoms for hemorrhagic, esterase and proteolytic activities Biochem. Pharmac. 20, 1549-l 5561 Homma M.. Kubota F.. Nikai T. and Sueihara H. (1980) Pathologic changes produced by loo-pacer snake venom and its purified proteinases; with special reference to hemorrhagic lesion. Kitakanto Iguku 30, 485494. Kondo H., Kondo S., Ikezawa H., Murata R. and Oshaka A. (1960) Studies on the quantitative method for the determination of hemorrhagic activity of Habu snake venom. Jap. J. med. Sri. Biol. 13, 43-51. Litchfield J. R. and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96, 99-113. Morris 0. L. (1948) Quantitative determination of carbohydrates with Dreywoods Anthrone reagent. Science 107, 254-255. Murata Y., Satake M. and Suzuki T. (1963) Studies on snake venom. Distribution of proteinase activities among Japanese and Formosan snake venoms. J. Biochem. 53,
431437. Nikai T., Sugihara H. and Tanaka T. (1977) Enzymochemical studies on snake Venoms. II. Purification of lethal protein Ac,-proteinase in the venom of Agkistrodon acutus. Yakugaku Zasshi 97, 507-514. Nikai T., Ishizaki H., Tu A. T. and Sugihara H. (1982) Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom. Comp. Biochem. Physiol.
72C, 103-106. Sugihara H., Nikai T., Kawaguchi T. and Tanaka T. (1978) Enzymochemical studies on snake venoms. IV. Purification of lethal protein AC,-proteinase in the venom of Agkbtrodon acutus. Yakugaku Zasshi 98, 1523-1529. Sugihara H., Nikai T., Umeda H. and Tanaka T. (1979) Enzymochemical studies on snake venoms. V. Purification of lethal protein AC,-proteinase in the venom of Agklrtrodon acutus. Yakugaku Zasshi 99, 1161-l 167. Tu A. T., Stermitz J. and Ishizaki H. (1980) Comparative study of pit viper venoms of Genera Trimeresurus from Asia and Bothrops from America: An immunological and isotachophoretic study. Comp. Biochem. Physiol. 66B, 249-254.