ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 298, No. 1, October, pp. 135-142, 1992

isolation and Characterization of Basic Myotoxic Phospholipases A2 from Bothrops godmani (Godman’s Pit Viper) Snake Venom Cecilia Diaz,*,t”

Jo& Maria Gutihrrez,*

and Bruno Lomonte*

*Institute Clodomiro Picado, Facultad de Microbiologia; and TDepartamento Escuela de Medicina, Universidad de Costa Rica, San Jo&, Costa Rica

Received December

LO, 1991, and in revised form, May 15,1992

Two basic myotoxic phospholipases A2 were purified to homogeneity from the venom of Bothrops godmani from Costa Rica by ion-exchange chromatography on CM-Sephadex. They have molecular weights of 14,300 (myotoxin I) and 13,400 (myotoxin II) and isoelectric points of 8.2 (myotoxin I) and 8.9 (myotoxin II). They behave as amphiphilic proteins in charge-shift electrophoresis and have similar amino acid compositions. Both toxins induce drastic myotoxic effects when injected in the gastrocnemius muscle of mice and induce release of peroxidase trapped in negatively charged liposomes. In addition, myotoxin I has high phospholipase AZ activity and is anticoagulant at doses higher than 0.3 pg/ml, whereas myotoxin II has a very low phospholipase AZ activity and exerts anticoagulant effect only at concentrations higher than 50 rg/ml. Immunochemical data indicate that both toxins are immunologically related to Bothrops asper myotoxins, although B. godmani myotoxin II gives a stronger cross-reactivity when tested with antisera raised against B. asper myotoxins I and II. 0 1992

Academic

Press,

de Fisiologia,

Inc.

Snake bite envenomations in Latin America are caused mainly by species of the genus Bothrops (1,2). In addition to systemic alterations, these envenomations are characterized by prominent local tissue damage due to myonecrosis, hemorrhage, and edema (1, 3). Acute muscle damage induced by these venoms is due mainly to basic myotoxic phospholipases AZ which initially affect the plasma membrane of muscle cells (3-5). Myotoxic phospholipases A, have been purified from the venoms of Bothrops asper (3, 6, 7), B. atrox (8), B. i To whom correspondence should be addressed at Institute Clodomiro Picado, Facuhad de Microbiologia, Universidad de Costa Rica, San Jose, Costa Rica. Fax: (506) 24 93 67. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

moojeni (8,9), B. nummifer (5, lo), B. jaruracussu (9, ll), B. neuwiedi (9), and B. pradoi (9). Immunochemical studies have revealed the presence of similar proteins in the venoms of other Central and South American Bothrops (12, 13). Some of these toxins have relatively high phospholipase A2 activity, whereas others have extremely low enzymatic action, although all of them exert drastic myotoxicity. Two basic myotoxins have been isolated from the venom of B. godmani, a terrestrial species distributed at high and intermediate elevations in Central America (2, 14). In this work we describe the biochemical characteristics of the toxins, as well as some of their pharmacological and immunological properties. MATERIALS

AND

METHODS

Materials. Venom was obtained from more than 60 adult specimens of B. godmani collected in Las Nubes de Coronado (San Jose, Costa Rica). After lyophilization, venom was kept at -30°C. The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): liposome kits, phospholipids, horseradish peroxidase, thrombin, thromboplastin, and kits for determination of creatine kinase. Reagents used in isoelectric focusing were from Pharmacia. Isolation of myotorins. Samples (250 mg) of crude venom were dissolved in 7.0 ml of 0.05 M Tris, 0.1 M KCI, pH 7.0, buffer and centrifuged at 5OOgfor 10 min. The supernatant was loaded on a CM-Sephadex C25 column (25 X 2 cm) equilibrated with the same buffer. Elution was carried out with a continuous KC1 concentration gradient (0.1 to 0.75 M). Active fractions were rechromatographed under identical conditions. Myotoxic and phospholipase A2 activities of the tubes were determined as described below. Active fractions were dialyzed against distilled water and lyophilized. Homogeneity. Purified proteins were analyzed by SDS’-polyacrylamide gel electrophoresis (15), under both reducing and nonreducing conditions, on 12% gels, as well as by polyacrylamide gel electrophoresis

’ Abbreviations used: SDS, sodium dodecyl sulfate; PBS, phosphatebuffered saline; CTAB, cetyltrimethylammonium bromide; PAGE, polyacrylamide gel electrophoresis. 135

Inc. reserved.

136

DiAZ,

GUTIERREZ,

for basic proteins in the presence of urea (16). Gels were stained with Coomassie brilliant blue R-250. The isoelectric points of the proteins were esZsoelectric focusing. timated by isoelectric focusing in 5 mm dried and rehydrated immobilized pH gradient gels (Pharmacia-LKB Biotechnology). Gels were prepared with immobiline solutions to give a pH range of 6.0 to 10.0. After polymerization, gels were dried and stored. Rehydration was carried out in 8 M urea, 50 mM dithiothreitol, 2% Triton X-100 solution containing ampholytes (pH range 3.0 to 10.0) (17). Isoelectric focusing was run in oil at 4°C and 1500 V for 8-10 h. Then, gels were fixed overnight in 10% trichloroacetic acid and stained with Violet 17 (Serva) (18). Protein samples (1-2 nmol) were hydrolyzed Amino acid composition. with 6 N HCl in sealed vials for either 20 or 70 h at 110°C under vacuum. Amino acid compositions were determined after HPLC on a Beckman 6300 amino acid analyzer (Beckman, CA). Tryptophan was estimated after p-toluenesulfonic acid hydrolysis of the samples (19). Efiectof toxins on liposomes. Negatively charged liposomes (1,2-diacyl-sn-glycero-3-phosphorylcholine, 63 pmol; diacetyl phosphate, 18 pmol; cholesterol, 9 pmol; kit L-4262, Sigma) were prepared by sonication in a 0.5 mg/ml horseradish peroxidase solution. Then, after 2 h of incubation at 3’7”C, liposomes were gel filtered on a Sepharose 6B column equilibrated with 0.145 M NaCl-KC1 solution. The effect of myotoxins on liposomes was studied in microtiter plates by incubating 20 ~1 of liposome suspension and 20 pl of solutions of varying concentrations of myotoxins dissolved in PBS, pH 7.2, for 30 min at 37°C. Then, 40 pl of peroxidase substrate (2.5 mM 5-aminosalycilic acid, 0.025% HsOx, pH 6.0) was added and the color reaction was stopped with 6 N HeSOl. Absorbances at 492 nm were recorded in a microplate reader. In order to determine the role of phospholipase Az activity in liposome disruption, similar experiments were carried out in a 0.14 M NaCl, 0.01 M Tris, pH 7.2, buffer containing either 1.6 mM CaClx or 1.6 mM EDTA. Chromatographic fractions were tested Phospholipase As act&y. for phospholipase Ax activity by using egg yolk as substrate, as described by Lomonte et al. (8). After reaction, free fatty acids were extracted and titrated according to Dole (20). Enzymatic activity of purified myotoxins was determined by titration with a Radiometer PHM 82 pH meter, using 1,2-diacyl-sn-glycero-3-phosphorylcholine as substrate, in a reaction mixture containing Triton X-100 (2:l molar ratio of Triton XlOO:phospholipid), as described by Aird and Kaiser (21). In order to determine substrate specificity of myotoxin I, various amounts of 1,2-diacyl-sn-glycero-3-phosphorylcholine, 1,2-diacyl-snglycero-3-phosphorylethanolamine, and 1,2-diacyl-sn-glycero-3-phosphorylserine were incubated with myotoxin (4.5 pg/ml) in 0.1 M TrisHCl, 10 mM CaCl,, 1% Triton X-100, pH 8.5. Incubations were carried out for 15 min at 37°C and free fatty acids were then extracted and titrated according to Dole (20). Myotoxic actiuity. White Webster mice (18-20 g) were injected i.m. in the right gastrocnemius with 100 pg of each myotoxin dissolved in 50 ~1 of PBS. Controls received 50 ~1 of PBS. Mice were bled from the tail at 1, 3, and 6 h and blood was collected in heparinized capillary tubes and centrifuged. The creatine kinase (EC 2.7.3.2) activity of plasma was determined by using Sigma Kit 520. Creatine kinase activity was expressed in units per milliliter, with one unit resulting in the phosphorylation of 1 nmol of creatine per minute at 25’C. Mice were sacrificed by cervical dislocation 7 h after toxin injection and samples of injected muscle were taken and processed routinely for embedding in Spurr resin in order to have a histological assessment of myotoxicity. Sheep platelet-poor plasma was prepared by Anticoagulant activity. centrifuging titrated blood twice at 1OOOgat 5°C. For the assay, 500 nl of platelet-poor plasma was incubated with 100 ~1 of various toxin concentrations dissolved in PBS. Incubations were carried out for 10 min at 37”C, 100 ~1 of 0.25 M CaCl, was added, and the coagulation time was recorded. In control tubes plasma was incubated with PBS instead of toxins. Observations were carried out for a maximum period of 60 min. The effect of myotoxins on prothrombin time was assessed by incubating various amounts of toxins with 200 ~1 of 0.05 M Tris, 0.1 M NaCl,

AND

LOMONTE

0.25 M CaCl,, pH 7.5, buffer containing thromboplastin. Control tubes contained PBS instead of toxin. Incubations were carried out at 37°C for 30 min, 100 ~1 of plasma was added, and the coagulation time was recorded. Thrombin time was determined by incubating 100 ~1 platelet-poor plasma with 100 ~1 of 0.05 M Tris, 0.1 M NaCl, pH 7.5, buffer for 30 min at 37°C. Then, clotting was induced by adding 50 pl thrombin (0.05 unit). The effect of myotoxins on thrombin time was tested by incubating platelet-poor plasma with various amounts of toxin for 30 min at 37°C before the addition of thrombin (22). The effect of 1,2-diacyl-sn-glycero-3-phosphorylserine on the anticoagulant activity of myotoxins was assessed by incubating 400 pl of platelet-poor plasma with 100 ~1 of PBS containing 10 pg of toxin for 10 min at 37°C. Then, 100 ~1 of 0.25 M CaCl,, together with 50 ~1 of phospholipid (500 to 0.12 FM), was added and clotting time was determined. The procedure of Helenius and Simons Charge-shift electrophoresk. (23) was carried out with some modifications (24), in order to determine if the myotoxins behave as amphiphilic proteins. Briefly, electrophoresis of myotoxins was performed in 1% agarose gels in three different systems: (i) 0.05 M glycine-NaOH, 0.1 M NaCl (pH 9.0), containing 0.5% Triton X-100; (ii) the same buffer but containing in addition 0.05% of the cationic detergent cetyltrimethylammonium bromide (CTAB); and (iii) the same buffer as (i) but containing also 0.25% of the anionic detergent deoxycholate. Electrophoreses were run at 48 mA for 50 V-h. Ovalbumin was used as nonamphiphilic protein control. Purified myotoxins were tested by Zmmurwchemical cross-reactivity. gel immunodiffusion (25) against a rabbit antiserum to B. asper myotoxins I and II (12) and equine polyvalent antivenom (26), using B. asper myotoxin II as control. The binding of antibodies to purified myotoxins and crude venoms was tested by enzyme immunoassay. Wells of microtiter plates were coated overnight at 22-25°C with 0.4 pg of each toxin (or venom) dissolved in 100 ~1 of 0.1 M Tris, 0.15 M NaCl, pH 9.0, buffer. After five washings with 0.05 M Tris, 0.15 M NaCl, 1 mM MgCls, pH 7.4, buffer, antiserum was added at various dilutions starting with 1:50 (in the test using rabbit antiserum against B. usper myotoxin II), and 1:200 (polyvalent antivenom). Incubations were carried out for 2 h at 22-25°C and the binding of antibodies was detected with peroxidase-protein A conjugate using 5-aminosalycilic acid-H,02 as substrate. Absorbances were read at 495 nm in a microplate reader. Toxins purified from B. godmani venom and B. asper myotoxin I were tested by gel immunodiffusion and enzyme immunoassay using a rabbit antiserum against B. godmcmi myotoxin (first basic fraction, hereby called myotoxin I). For the enzyme immunoassay, wells of microtiter plates were coated with 0.4 pg of each toxin and the test was carried out as described above, using various dilutions of antiserum starting with 1:50. Lethality. Groups of four mice (16-18 g) were injected iv. with 200 ~1 of various toxin concentrations prepared in phosphate-buffered saline solution, pH 7.2. Deaths were recorded for 48 h and LDm was estimated by using the Spearman-Karber method (27,28). Edema. The method of Yamakawa et al. (29) was used. Groups of four mice (18-20 g) were injected S.C. in the right foot pad with 50 ~1 of solutions containing different amounts of toxin (5-40 wg), whereas the left foot pad was injected with 50 ~1 of PBS. Mice were killed by cervical dislocation 5 h after injection and both feet were cut and weighed. Edema was expressed as the percentage increase in weight of the right foot compared to that of the left one. RESULTS

Isolation and characterization of myotoxins. Ion-exchange chromatographic fractionation of B. godmani venom gave several peaks with myotoxic and phospholipase A2 activities (Fig. 1). The highest phospholipase A2 activity is associated with the acidic peak and the first

PHOSPHOLIPASES

Ax IN Bothrops

godmani

137

VENOM

a very low activity (2 bmol fatty acid. mg-’ min-l). Myotoxin I had the following substrate preference: l,2diacyl-sn-glycero-3-phosphorylcholine > 1,2-diacyl-snglycero-3-phosphorylethanolamine > 1,2-diacyl-sn-glycero3-phosphorylserine. After i.m. injection both myotoxins Myotoxic activity. induced a rapid and drastic increase in plasma creatine kinase, which is a specific marker for muscle damage. Maximum levels were observed at 3 h, and decreased afterward (Fig. 3). Histological observations in samples obtained 7 h after toxin injection corroborated myotoxicity. Necrotic fibers were abundant and contained clumped and dense masses of myofibrils (Fig. 4). Effectsof myotoxins on liposomes. Both myotoxins induced a dose-dependent release of peroxidase trapped in negatively charged liposomes (Fig. 5), with no significant difference between the activities of these toxins (P > 0.1). In the case of myotoxin I, the liposome-disrupting effect was higher (P < 0.005) when calcium was present in the medium than when calcium was excluded and EDTA added (Fig. 6A). In contrast, myotoxin II disrupted liposomes to a similar extent in both the presence and the absence of calcium (P > 0.1) (Fig. 6B). Figure 7 shows that the recalAnticoagulant activity. cification time of sheep platelet-poor plasma was indefinitely prolonged after incubation with myotoxin I at concentrations of 0.3 pug/ml or higher. On the other hand, myotoxin II prolonged recalcification time at much higher concentration (50 pg/ml). l

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470 370 270

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of Bothrops godmani venom by ion-exchange FIG. 1. Fractionation chromatography on CM-Sephadex C-25. Samples (250 mg) of crude venom dissolved in 7.0 ml of 0.05 M Tris, 0.1 M KC1 (pH 7.0) buffer were loaded on the column equilibrated with the same buffer. Elution was carried out with a continuous KC1 concentration gradient (0.1 to 0.75 M). Open circles show protein concentration (absorbance at 280 nm); (A) filled circles show phospholipase Ax activity (peq NaOH . mini); and (B) filled diamonds show myotoxic activity (plasma creatine kinase levels Units/ml).

basic myotoxic peak, whereas myotoxic activity is present in the most basic peaks. After recycling of each of the basic fractions on the same CM-Sephadex column, single symmetrical peaks corresponding to myotoxins I and II were obtained. Both proteins migrated as single bands on SDS-PAGE and urea-PAGE (Fig. 2). Amino acid compositions are presented in Table I. Molecular weights of these myotoxins are 15,000 (myotoxin I) and 14,500 (myotoxin II) as estimated by SDS-PAGE and 14,300 (myotoxin I) and 13,400 (myotoxin II) as estimated by amino acid analysis. Isoelectric points of the toxins were 8.2 (myotoxin I) and 8.9 (myotoxin II). Both proteins behaved as amphiphilic molecules in charge-shift electrophoresis, as evidenced by their different migration depending upon the charge of the detergent in the gels. Ovalbumin migrated in a similar way in the three systems tested, as expected for a nonamphiphilic protein (Table II). Myotoxin I displayed a relPhospholipase AZ activity. atively high activity of 765 pmol fatty acid * mg-’ . min-’ when 1,2-diacyl-sn-glycero-3-phosphorylcholine was used as substrate. In the same assay system, myotoxin II showed

123 FIG. 2. Electrophoretic analyses of Bothrops godmani myotoxins. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%). Samples were reduced with 2-mercaptoethanol at 95°C. Lane 1, mol. wt. markers (kDa) (phosphorylase b, 94; albumin, 67; ovalbumin, 43; carbonic anhydrase, 30, trypsin inhibitor, 20.1; a-lactalbumin, 14.4); lane 2, B. godmani myotoxin I, 4 pg; lane 3, B. godmani myotoxin II, 4 pg; lane 4, B. godmani venom, 20 pg. (B) Urea-polyacrylamide gel electrophoresis (12%) for basic proteins at pH 4.5. Cathode is at bottom. Lane 1, B. godmani myotoxin II, 10 pg; lane 2,B. godmani myotoxin I, 10 fig; lane 3, B. godmuni venom, 50 pg. Gels were stained with Coomassie blue R-250.

138

DiAZ, TABLE

Amino

Acid

Compositions

of

Myotoxin

Amino acid ASP Thr Ser Glu Pro GUY Ala CYS Val Met Ile Leu Tyr Phe His LYS Trp Arg Total residues: Formula weight:

Mol amino acid/m01 protein 13.92 6.79 2.85 8.12 5.22 10.12 7.15 13.66 6.07 3.05 4.67 2.62 7.78 7.25 2.00 14.08 3.25 5.01

GUTIERREZ,

AND

LOMONTE TABLE

I

Myotoxin

I

Nearest integer 14 7 3 8 5 10 7 14 6 3 5 3 8 7 2 14 3 5 124 14,300

Mol amino acid/m01 protein 15.24 6.06 6.11 6.33 5.90 6.89 5.98 13.54 4.87 3.09 4.08 7.06 7.86 2.08 1.00 14.46 3.80 3.01

II

in Charge-Shift Migration of B. godmani Myotoxins Electrophoresis Agarose Gels Containing Triton X-100, Triton Plus CTAB, or Triton Plus Deoxycholate

Bothrops godmani Myotoxins II

Nearest integer 15 6 6 6 6 7 6 14 5 3 4 7 8 2 1 14 4 3 117 13,400

In addition, myotoxin I prolonged prothrombin time (Fig. 8), but had no effect on thrombin time. The negatively charged phospholipid 1,2-diacyl-sn-glycero-3phosphorylserine prevented, in a dose-dependent manner, the anticoagulant effect of myotoxin I (Fig. 9). Otherpharmacological activities. Both toxins induced a mild edema when tested by the foot-pad assay. Injections of 40 /*g caused an edema of 20 f 1.5% (myotoxin I) and 30 k 3.0% (myotoxin II) 5 h after injection. Myotoxin II had an i.v. LD5,-, of 4.2 pg/g (95% confidence limits: 3.6 to 4.9 pg/g), whereas myotoxin I did not induce lethality at a dose of 5 pg/g. Immunochemical characterization. When both toxins were tested by immunodiffusion with rabbit antisera raised against B. asper myotoxins I and II, only B. godmani myotoxin II formed a precipitin band, having a pattern of partial identity with both myotoxins of B. asper. Enzyme immunoassays demonstrated that a polyclonal rabbit antiserum against B. asper myotoxin II recognized both myotoxins of B. godmani venom. However, the reaction was stronger against myotoxin II than against myotoxin I (Table III). Commercially available polyvalent antivenom reacted against both B. godmani myotoxins when tested by ELISA, although no reaction was observed by gel immunodiffusion. On the other hand, an antiserum raised in rabbits against B. godmani myotoxin I recognized its homologous antigen and cross-reacted with B. asper myotoxin II and B. godmani myotoxin II (Table III).

Detergents in the gel Triton X-100 and CTAB Triton X-100 Triton and deoxycholate

Myotoxin

I

Myotoxin

II

Ovalbumin

6.7 + 1.7 (C)” 3.0 + 0.1 (C)

8.3 + 1.3 (C) 4.0 k 0.1 (C)

19.0 + 0.8 (A) 18.0 f 0.8 (A)

12.8 + 1.3 (A)

13.8 + 1.9 (A)

16.0 & 1.0 (A)

Note. Electrophoresis was carried out at 48 mA for 50 V-h. Results are presented as means + SE (rz = 3). ’ Migration of each toxin toward the anode (A) or the cathode (C) is expressed in mm.

DISCUSSION Two myotoxic phospholipases A2 were isolated from the venom of B. godmani. Both are basic proteins with similar molecular weights and amino acid compositions, behaving as amphiphilic proteins in charge-shift electrophoresis. Despite differences in phospholipase AZ and anticoagulant activities, immunochemical observations suggest that both myotoxins belong to a group of basic myotoxins with phospholipase A2 structure and similar amino acid compositions isolated from the venoms of B. asper (6,7,30), B. moojeni (8,9), B. atrox (8), B. nummifer (5, lo), and B. jararacussu (11,31). Similar toxins are also present in several other Central and South American Bothrops venoms (9,12, 13). Within this “family” of myotoxins, sequence studies have revealed the existence of

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FIG. 3. Changes in plasma creatine kinase (CK) levels after i.m. injection of B. godmani myotoxins. One hundred micrograms of the toxins (in 100 ~1 of PBS) was injected i.m. into the gastrocnemius of mice. At various time intervals mice were bled and plasma CK levels determined. CK activity is expressed in units/ml, with one unit resulting in the phosphorylation of 1 nmol of creatine per minute at 25’C. Results are presented as means f SE (n = 4). Open circles, myotoxin I; filled circles, myotoxin II. Mice injected with 100 ~1 PBS had CK values of 40 + 4.5 at 3 h.

PHOSPHOLIPASES

A2 IN Bothrops

godmani

VENOM

139

FIG. 4. Light micrographs of mouse gastrocnemius muscle 7 h after i.m. injection of 100 pg of B. godmani myotoxin I (A) and B. gcldmani myotoxin II (B). Note widespread myonecrosis with muscle fibers showing conspicuous myofibrillar disorganization indicative of muscle cell I death. A mild inflammatory infiltrate is also observed. Bars represent 50 pm.

140

DiAZ.

GUTIfiRREZ.

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LOMONTE 70 60

?

EM ‘Z p .t: 8

30-/ 20--

+

lo-

04

:

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I 10

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1000

01

/ ?-m-?-AIA-A: 10

100

Toxin &/ml)

-A 1000

.A

10000

Toxin (rig/ml)

FIG. 5. Release of peroxidase trapped in negatively charged liposomes incubated with B. godmani myotoxins I and II. Release is expressed as a percentage, with 100% as the peroxidase release from liposomes incubated with 0.2% Triton X-100. Open circles, B. godmani myotoxin I; filled circles, El. godmani myotoxin II. Results are presented as means + SE (n = 6).

FIG. 7. Coagulation time of sheep platelet-poor plasma recalcified after incubation with B. godmuni myotoxin I (filled circles) and myotoxin II (open triangles). Plasma was incubated for 10 min at 37°C with various toxin concentrations. Then, CaCl, was added and clotting time recorded. Observations were carried out for a maximum period of 60 min. Results are presented as means k SE (n = 6).

both aspartate-49 and lysine-49 variants (7,32), the latter being devoid of or having extremely low phospholipase Az activity (32-35). At present it is not known if B. godmani myotoxin II is a lysine-49 variant, as it has very low, but detectable, enzymatic activity. On the other hand,

B. godmani myotoxin I has high phospholipase AZ activity (765 pmol fatty acid * mg-’ . min-‘) when compared to the catalytically active basic enzymes isolated from other Bothrops venoms, whose activities range between 2 and 430 pmol fatty acid. mg-’ . min-’ (7,8, 31). The two toxins isolated from B. godmani venom induce acute muscle damage upon i.m. injection in mice, showing a similar time course of creatine kinase increment in plasma and inducing identical histological alterations in skeletal muscle tissue. On the basis of these observations, it is likely that the pathogenesis of myonecrosis caused by these toxins is similar to that induced by other Bothrops myotoxins (3-5, 31). Since B. godmani myotoxin II has

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FIG. 6. Effect of B. godmani myotoxin I (A) and myotoxin II (B) on the release of peroxidase trapped in negatively charged liposomes, in media containing either CaC& (open circles) or EDTA (filled circles). Peroxidase release is expressed as a percentage (see legend to Fig. 5). Results are presented as means +- SE (n = 3).

FIG. 8. Effect of B. godmani myotoxin I on prothrombin time (filled circles) and thrombin time (open circles) clotting assays. For prothrombin time experiments, various concentrations of toxin were incubated with thromboplastin and CaCl, at 37°C for 30 min. Then, plasma was added and coagulation time recorded. The effect of myotoxin I on thrombin time was determined by incubating platelet-poor plasma with various toxin concentrations for 30 min at 37’C. After incubation, thrombin was added and clotting time recorded. Results are presented as means f SE (n = 3).

PHOSPHOLIPASES

As IN Bothrops

very low enzymatic activity, it is suggested that myotoxicity does not depend on enzymatic phospholipid degradation, but probably on a molecular region different from the catalytic site, as has been suggested for a variety of pharmacological effects induced by phospholipases AZ (3, 36, 37). Both myotoxins disrupted negatively charged liposomes, as has been also found for other myotoxins isolated from the venoms of B. asper, B. atror, and B. moojeni (24). However, the dependence of the liposomal-disrupting effect on phospholipid hydrolysis differs between the toxins: myotoxin II exerted its activity even under conditions where catalysis is inhibited. In contrast, there was a significant reduction in the liposomal-disrupting effect of myotoxin I when calcium was eliminated and EDTA added to the incubation medium. Thus, the membranedisrupting activity of myotoxin I probably depends, at least partially, on the ability of the toxin to hydrolyze bilayer phospholipids. Many phospholipases AZ isolated from snake venoms are anticoagulant. In this regard, Bothrops myotoxins can be divided into two groups: B. asper myotoxins I and III, B. atrox myotoxin, B. godmani myotoxin I, and bothropstoxin II from B. jararacussu are strong anticoagulants, whereas B. asper myotoxin II, B. moojeni myotoxins I and II, and B. nummifer myotoxin lack this effect (7, 8, 10, 30, 31, 38). B. godmani myotoxin II occupies an intermediate position, as it has very low phospholipase AZ activity and prolongs recalcification time only at high doses. Our results suggest that the anticoagulant effect of myotoxic phospholipases AZ from Bothrops venoms is dependent on enzymatic degradation of phospholipids necessary for the adherence and activation of coagulation protein factors. This hypothesis is supported by the observation that myotoxins prolong both recalcification time and prothrombin time, but not thrombin time, since only

40

godmani

141

VENOM TABLE

III

Cross-Reactivity of Antisera against Myotoxins and Polyvalent Antivenom with B. godmani and B. asper Myotoxins and Crude Venoms

B. B. B. B. B.

asper MT-II godmani MT-I godmani MT-II asper venom godmani venom

Anti-B. asper myotoxin II

Anti-B. godmani myotoxin I

1.899 + 0.009” 1.054 k 0.115 1.514 + 0.028 NTb NT

0.867 * 0.020 1.594 * 0.144 1.063 + 0.084 NT NT

Polyvalent antivenom

0.327 0.291 0.837 0.691

NT ? 0.001 2 0.002 k 0.020 f 0.023

’ Absorbance at 492 nm using antiserum dilutions of 1:200 (anti-B. asper MT-II), 1:400 (anti-B. godmani MT-I), and 1:1600 (polyvalent antivenom). Results are presented as means -+ SD (n = 2). Negative controls using normal rabbit serum gave absorbances of 0.244 ? 0.010 (anti-B. asper MT-II test) (n = 6) and 0.052 + 0.020 (anti-B. godmani (MT-I test) (n = 6). Controls using normal horse serum gave absorbances of 0.092 + 0.020 (n = 8). b NT, not tested.

the former two depend on plasma phospholipids (22). Moreover, the fact that addition of 1,2-diacyl-sn-glycero3-phosphorylserine prevented the anticoagulant effect of myotoxin I on platelet-poor plasma further strengthens this conclusion. Toxins with extremely low phospholipase A2 activity probably bind, but do not hydrolyze to a significant extent, plasma phospholipids and, therefore, do not affect their role in coagulation. All Bothrops myotoxins tested so far are amphiphilic proteins able to disorganize liposomal membranes (5,24,31). Therefore, it is suggested that anticoagulability induced by these toxins depends not only on phospholipase A2 activity but also on the ability to penetrate membranes. Thus, although there are controversial hypotheses concerning the role of enzymatic activity in the anticoagulant effect of phospholipases (see Kini and Evans (36) and Rosenberg (37) for reviews), in the case of Bothrops myotoxins observations clearly support the hypothesis of Verheij et al. (39) that anticoagulation by phospholipases AZ depends on both penetration into phospholipid bilayer and enzymatic degradation of phospholipids. ACKNOWLEDGMENTS

0 1

10 Phospholipid

100

1000

(nanomoles)

FIG. 9. Effect of 1,2-diacyl-sn-glycero-3-phosphorylserine on the anticoagulant activity of B. godmani rnyotoxin I. Platelet-poor plasma (400 fil) was incubated with 100 ~1 of PBS containing 10 pg of toxin for 10 min. Then, CaCl, andvarious amounts of 1,2-diacyl-sn-glycero-3-phosphorylserine were added and clotting times recorded. Results are presented as means + SE (n = 6).

The authors thank Dr. I. I. Kaiser for phospholipase Az activity determinations, Dr. J. P. Rosso and Dr. 0. Vargas for the amino acid composition determinations, and Mr. J. NCiiez for his valuable collaboration in the laboratory and photographic work. This study was supported by Vicerrectoria de Investigacibn, Universidad de Costa Rica (Project 741-90-044). J. M. Gutierrez and B. Lomonte are recipients of a research career award from the Costa Rican National Scientific and Technological Research Council (CONICIT). This work was done as partial fulfillment of the requirements for the M.Sc. degree for Cecilia Diaz-Oreiro at the University of Costa Rica.

142

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