Toska~, Vd . 30, No . 8, pp . 85~864, 1992. Printed in Great B~trin.

0041-0101/92 SS.00 + .00 © 1992 Per~emon Pten Ltd

PURIFICATION AND CHARACTERIZATION OF TWO FIBRINOLYTIC ENZYMES FROM BOTHROPS JARARACA (JARARACA) VENOM Mnsucl MNeuYnMw,' MnsnFUICO Sucncl,'

ETSUO YosIImn,' HISnsIIi MIxnRn' and NnxnrnKn2

NOBUYOSHI

Department of Physiology, Miya7aki Medical College, Kiyotake, Miyazaki, Japan; and Department of Nutrition and Food Science, Okayama Prefectural College, Okayama, Okayama, Japan (Received 15 January 1992; accepted 21 February 1992)

M. MnRUYnMn, M. Sucra, E. Yosi->m~, H. Mlxwxn and N. NAx~JnKA. Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom. Toxicon 30, 853-864, 1992 .-Two fibrinolytic enzymes, jararafibrase I and jararafibrase II, were purified from Bothrops jararaca venom. The purified jararafibrase I and jararafibrase II ran as single protein bands on analytical polyacrylamide gel electrophoresis and had mol. wts of 47,000 f 2000 and 21,400 f 500, respectively, by SDS-polyacrylamide gel electrophoresis. The isoelectric points of jararafibrase I and jararafibrase II were 4.6 and 6.5, respectively . The specific activities of jararafibrase I and jararafibrase II were 2.2 units/mg protein and 6.3 units/mg protein, respectively . Both enzymes exhibited no detectable plasminogen activating activity . The activity of the enzymes was completely inhibited by 1,10-phenanthroline and ethylenediaminetetraacetate, suggesting that both enzymes were metalloproteinases . Jararafibrase I and jararafibrase II had single-chain protein compositions, and the amino acid sequence up to the 49th amino acid from the NHZterminal of jararafibrase II was: Leu-Pro~luHis~ln-Arg-Tyr-Ile~lu-Leu-Phe-Ile-Val-Val-Asp-His-Gly-Met-Phe Met-Lys-Tyr-Asn~ly-Asn-Ser-Asp- Lys-Ile-Arg-Arg-Arg-Ile-His-GlnMet-Val-Asn-Ile-Met-Lys-X-Ala-Tyr-Arg-Tyr-Leu-Tyr-IIe~X =not confirmed) .

INTRODUCTION

is well known that snake venoms, especially from the Crotalidae and Viperidae snakes, possess factors which affect the blood clotting system of victims and provoke severe coagulopathy with unclottable blood. Although the main cause of such coagulopathy involves a coagulant activity, fibrinolytic activity may also play an important role in the manifestation of systemic coagulopathy in envenomed patients, since most fibrinolytic enzymes have a fibrinogenolytic activity . Apart from the effect of the fibrinolytic system in dissolving fibrin clots in the circulatory system, the action of the fibrinolytic system can be that of a trigger enzyme in the local inflammatory process (MORIMOTO et al., 1991) and tissue matrix breakdown (BocENMwxrt and JoNas, 1983). The fibrinolytic activity of snake IT

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FIG . I . ELUTION PROFILE OF BOthrOpS jClraraCa CRUDE VENOM BY SEPHADEX G-7S MOLECULAR SIEVE CHROMATOGRAPHY . Four milliliters of Bothrops jararaca venom (25 mg/ml) was applied on a column (2.S x 70 em) of Sephadex G-7S equilibrated with 10 mM Tris-HCl buffer containing 10 mM CaCI=, 0.1 S M NaCI, pH 8 .0. The flow rate was 4 .1 ml/cm=/hr . Fractions I and II were collected as indicated by the horizontal bars . --, Protein concentration ; -~-, fibrinolytic activity ; -/-,clotting activity .

venoms may thus have an accelerative effect on the inflammatory process as well as on haemorrhage and necrosis occurring in the bitten area . Some fibrinolytic enzymes purified from snake venoms display a haemorrhagic activity (IMAI et al ., 1989; SExoGUCI-n et al., 1989; HUANG et al., 1984 ; $IIGUR and SIIGUR, 1991) and some do not (ARAGON-ORTIZ and GUBENFSEK, 1987 ; WILLIS and Tu, 1988 ; CüEN et al., 1991). In South America, at least 90% of snake envenomation is caused by Bothrops species. We examined the fibrinolytic activity of nine Bothrops snake venoms, and found that all of them had fibrinolytic enzymes as well as coagulant enzymes (FURTADO et al., 1991). In the south-eastern area of Brazil, the most important species causing snake envenomation is Bothrops jararaca (MARUYAMA et al., 1990). We purified two fibrinolytic enzymes from B. jararaca venom and determined the NH Zterminal amino acid sequence up to the 49th amino acid of one of the two enzymes. Some of the enzymatic and physicochemical characteristic of the enzymes were also investigated .

MATERIALS AND METHODS Materials Sephadex G-75 (medium size), Sephadex G-7S (superfine), and Sephacryl 5-200 (high resolution) were obtained from Pharmacia Fine Chem . (Uppsala, Sweden) . Affi-Gel blue for dye ligand chromatography was purchased from Bio-ltad Lab . (Richmond, CA, U.S .A .) . Polyethyleneglycol (average mol . wt 20,000) and (pamidinophenyl) methanesulfonyl fluoride were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan) . Fibrinogen (bovine, 7S% clottable) was purchased from Miles Inc . (Kankakee, IL, U .S.A .) . Soybean trypsin inhibitor and N°P-tosyl-L-lysine chloromethyl ketone were purchased from Sigma Chemical Co. (St . Louis, MO, U .S .A .) . Dithiothreitol and I,10-phenanthroline were purchased from Nacalai Tesque, Inc . (Kyoto,

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Praotlon Number (4ml/Tube) FIG. 2. ELUTION PROFILE OF FRAC110N I BY AFFI-GEL BLUE Ctt1tOINATOGRAPHY. Fraction I (6 .6 ml, 60.7 mg) was applied to a column (1 .6 x l5 cm) of A~-Gel blue equilibrated with 10 mM Tris-HCI buffer containing 10 mM CaClz, pH 8.0. The column was washed with the same buffer and the clotting activity was eluted by the buffer containing 2 M NaCI . The flow rate was 10.2 ml/cm2/hr. Fraction Ia was collected as indicated by the horizontal bar. -, Protein concentration; -~-, fibrinolytic activity; -/-,clotting activity .

Japan). Elastatinal was purchased from Peptide Institute, Inc. (Osaka, Japan) . H-n-Val- Leu-Lys-pNA (s-2251) was purchased from Daüchi Pure Chemicals Co . Ltd (Tokyo, Japan) . Venom source A pool of lyophilized venom from adult specimens of B. jararaca maintained at the Laboratory of Herpetology, Instituto Butantan, Brazil, was used . Assay offibrinoiytic activity The fibrinolytic activity was measured using 0.6% bovine phu:minogen-rich fibrin plates or 0.6% bovine plasminogen-free fibrin plates (MORIMOTO et ai., 1991). Thirty microliters of sample was placed on the fibrin plates and the lysis areas were measured after incubation at 37°C for 18 hr. The specific activity was calculated from a standard curve for the lysis area with plasntin (Sigma) on plasminogen-free fibrin plates . One unit of fibrinolytic activity was defined as being of the same magnitude as the lytic activity of l casein unit (CU) of standard plasmin on a phlsminogen-free fibrin plate. For specific activity determinations, the protein concentration was estimated by the method of LOWRY et al. (1951) using bovine serum albumin as a standard . Assay ojciotting activity Two-hundred microliters of human citrated plasma was mixed with 50 pl of sample and the clotting time was recorded at 37°C . The clotting activity was expressed at l/clotting time (sec) x lOz. Plasminogen activating activity assay The plasminogen activating activity was determined according to the method of VERHEUEN et al . (1982). Nine hundred microliters of 0.1 M Tris-HCl buffer, pH 7.5, containing 0. l% Tween 20, 0.13 pM of human Glu-plasminogen, l0 mM CaCl r and various concentrations of samples were mixed with 100 pl of 3 mM 5-2251 and incubated at 37°C . The activity was measured by rewrding the colour development at 405 nm continuously, employing a phtsminogen-frce assay mixture as a reference. t>u n~e-o

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FIG. 3 . ELU77ON PROFILE OF FRACTION Ial HY SEPHACRYL 5-200 (HIGH RESOLUTION) AtOLECI7LAR SIEVE CFRtOMATOGRAPHY . Fraction Ial (0 .3 ttil, 1 .7 mg) was applied to a column (0 .9 x 45-cm) of Sephacryl 5-200 (high resolution) equilibrated with 10 mM Tris-HCl buffer containing 10 mM CaC12 , 0.75 M NaCI, pH 8.0 . The flow rate was 7 .5 ml/cm'/hr . Jararafibrase I was obtained by collection of the fractions indicated by the horizontal bar. -, Protein concentration ; -~-, fibrinolytic activity .

Haernorrhagic activity The haemorrhagc activity was determined by the method of THEAICSTON and REID (1983) . Fifty microliters of sample was injected intradermally into the dorsal skin of rats, and the area of haemorrhage was measured at 24 hr after the injection .

Molecular sieve colwtve chromatography A Sephadex G-75 (medium size) column (2 .5 x 70 cm) was equilibrated with 10 tnM Tris-HCI, IO mM CaC( Z , 0.15 M NaCI, pH 8.0, and 4 ml of B. jararaca venom solution (25 mg/ml) was applied. Elution was performed with the same buffer and fractions consisting of 4 ml/tube were collected . As mol . wt markets, bovine serum albumin (mol. wt 67,000, Gibco Laboratories, NY, U .S.A .), ovalbumin (mol . wt 45,000, Sigma Chem. Co .), achymotrypsinogen A (mol . wt 25,000, Sigma Chem . Co .) and cytochrome c (mol . wt 12,800, Sigma Chem. Co.) were used. A Sephadex G-75 (superfine) column (0.5 x 45 cm) was equilibrated with 10 mM Tris-HCI, 10 mM CaCIZ , 0 .75 M NaCI, pH 8 .0, and approximately 0 .3 ml of sample was applied to the column . Elution was performed with the same buffer and fractions of 0.5 ml/tube were collected . A Sephacryl 5-200 (high resolution) column (0.9 x 45 cm) was equilibrated with l0 mM Tris-HCI, 10 mM CaC12, 0.75 M NaCI, pH 8 .0, and approximately 0 .3 ml of sample was applied . Elution was performed with the same buffer and fractions of 0 .5 ml/tube were collected.

Affinity chromatography An A~-Gel blue gel column (1 .6 x 15 cm) was equilibrated with 10 mM Tris-HCI, 10 mM CaCIZ, pH 8.0, or 10 mM CaC1 2 solution . After application of the sample, the column was washed with the equilibration buffer or solution . Elution was perforated with 2 M NaCI, or a 0-2 M NaCI linear gradient, or a 0-20 mM Tris linear gradient . All purification procedures were carried out at 4°C .

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FIG. 4. ELUTION PROFILE OF FRACTION II BY AFFI-GEL BLUE CHROINATOGRAPHY. Fraction I (12.2 ml, 29.3 mg) was applied to a column (I .6 x 15 cm) of Affi-Gel blue equilibrated with l0 mM Tris-HCl buffer containing 10 mM CaClr pH 8.0 . The column was washed with the same buffer and the fibrinolytic activity was eluted with a 0-2 M NaCI linear gradient . The flow rate was 6.9 ml/ctn=/hr . Fractions Ila and Ilb were collected as indicated by the horizontal bars . -, Protein concentration; -~-, fibrinolytic activity; - --- -, NaCI gradient .

Polyacrylamide gel electrophoresis Analytical polyacrylamide gel electrophoresis (analytical PAGE) was performed aceording to the method of DAMS (1964), and SDS-PAGE was carried out by the method of LnE~atLI (1970), in the presence of 3 M urea . Prestained protein mol. wt standards (GIBCO BRL, Gaithersburg, MD, U.S.A.) were used for mol. wt estimation .

lsoeJectric focusing Isoelectric focusing was carried out on agarose gel plates according to the method of VP3rERBIItO (1972), employing carrier amphorite pH 4.0-7 .0 (Bie-Rad Lab., Richmond, CA, U.S .A.) .

Amino acid analysis Amino acid sequencing was performed with an automated gas-phase sequence analyzer (Applied Biosystems, model 470A), and PTH-amino acids were analyzed by HPLC employing an Ultrasphere ODS column (MARUYAINA et al., 1986).

Fibrinogen degradation tests Two hundred microliters of fibrinogen (2 mg/ml) was mixed with enzymes dissolved in 10 mM Tris-HCI, 10 mM CaC12 , pH 8.0, and the mixture was incubated at 37°C . Samples were withdrawn at various time intervals and subjected to SDS-PAGE under reduced conditions .

RESULTS

Four milliliters of B. jararaca venom solution (25 mg/ml) was applied to a column (2.5 x 70 cm) of Sephadex G-75 equilibrated with 10 mM Tris-HCI, 10 mM CaC12, 0.15 M NaCI, pH 8.0. A typical elution profile of the chromatography is shown in Fig. 1 . The

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FIG . S . ELUTION PROFILE OF FRACTION IIbI BY SEPHADEX G-7S (SUPERFINE) MOLECULAR SAVE CHROMATOGRAPHY .

Fraction IIbI (0.3 ml, 0 .61 mg) was applied to a column (0.9 x 4S crn) of Sephadez G-7S (superfine) equilibrated with 10 mM Tris-HCl buffer containing 10 mM CaCl z, 0 .75 M NaCI, pH 8 .0. The flow rate was S .6 ml/cmZ/hr. Jararafibrase II was obtained by collection of the fractions indicated by the horizontal bar . ----, Protein concentration ; -~-, fibrinolytic activity .

fibrinolytic activities were eluted in two peaks. The mol . wts of the first and the second peaks calculated in relation to standard marker proteins were approximately 58,000 and 18,000, respectively . Clotting activity was eluted in one peak at a mol. wt of about 60,000. The two fibrinolytic peaks, fractions I and II, were collected and concentrated using polyethyleneglycol . The fibrinolytic activity of the partially purified fractions I and II assayed in calcium-free condition was decreased to 44% and 70%,respectively, of the activities assayed with 10 mM CaCIZ. The concentrated fraction I (6 ml) was dialyzed against 10 mM Tris-HCI, 10 mM CaCIZ, pH 8.0, at 4°C overnight. The dialyzed material was applied to an Affi-Gel blue column (1.6 x 15 cm) equilibrated with 10 mM Tris-HCI, l0 mM CaCIZ, pH 8 .0. The column was then washed with the same buffer. As shown in Fig. 2, the nonadsorbed fractions were separated into two protein peaks and the fibrinolytic activity appeared to be coincident with the second protein peak . The clotting activity was strongly adsorbed and 2 M NaCI was necessary to elute the activity . The fibrinolytic fractions indicated in Fig. 2 were collected (fraction Ia) and concentrated using polyethyleneglycol . The concentrated fraction Ia (11 .3 ml) was dialyzed against 10 mM CaCIZ overnight and applied again to the A~-Gel blue column equilibrated with 10 mM CaCIZ and washed with the same solution . The nonadsorbed fractions displayed no fibrinolytic activity. Elution of fibrinolytic activity was performed with a linear gradient of 0-20 mM Tris-HCl buffer containing 10 mM CaCI Z, pH 8 .0. The fibrinolytic activity was eluted as a single peak at approximately 7 mM of Tris. The fibrinolytic activity peak was collected (fraction Ial) and concentrated using polyethyleneglycol followed by dialysis against IOmM

Fibrinolytic Enzymes from

859

Bothrops jararaca

TABLE I . SLJIONARY OF PURIPICAIION STEPS

Procedure Sephadex G-75 Affi-Gel blue (first step) Affi-Gel blue (second step) Sephacryl 5-200 Sephadex G-75 Affi-Gel blue (first step) Affi-Gel blue (second step) Sephadex G-75

Fraction

Protein (mg)

Activity (units)

Yield (%)

Spec act. (units/mg)

Crude venom I Ia Ial Jararafibrase I II IIb IIbI Jararafibrase II

100.0 60 .7 13 .3 3.40 1.52 29.3 3.59 1.22 0.35

240.0 80 .5 18 .5 3.76 3.36 136.6 31 .1 3.40 2.20

100.0 34 .2 7.9 1 .6 l .4 58 .1 13 .2 1 .4 0.9

2.4 1.3 1 .4 L1 2.2 4.6 8.7 2.8 6.3

Protein concentration was determined by the method of LowRY et al. (1951), using bovine serum albumin as a standard . One unit of fibrinolytic activity was defined as being the same as the activity of one CU of plasmin (Sigma chemical Co .) on a plasminogen-free fibrin plate.

Tris-HCI, 10 mM CaCIZ, 0.75 M NaCI, for 6 hr. Three hundred microliters of the dialyzed fraction Ial was applied to a Sephacryl S-200 column (0.9 x 45 cm) and was obtained as a fibrinolytic enzyme (jararafibrase I) (Fig. 3). The concentrated fraction II (12.2 ml) yielded by the first step of Sephadex G-75 gel chromatography was dialyzed against 10 mM Tris-HCI, 10 mM CaCl2, pH 8.0, and applied to an Affi-Gel blue column (1 .6 x 15 cm) equilibrated with the same buffer as for the dialysis . The column was washed with the same Tris buffer, and the fibrinolytic activity was eluted with a linear gradient of 0-2 M NaCI . As shown in Fig. 4, no fibrinolytic activity was found in the nonadsorbed fractions, while two fibrinolytic activity peaks were obtained by the elution with the 0-2 M NaCI gradient . The first peak was eluted by approximately 0.4 M NaCI, and the second peak was eluted by approximately 0.7 M NaCI . The second peak, fraction IIb, was collected and concentrated using polyethyleneglycol . The concentrated fraction IIb (6.9 ml) was dialyzed against 10 mM Tris-HCI, 10 mM CaCI Z, pH 8.0, for 6 hr and applied to the same Affi-Gel blue column . Elution of the fibrinolytic enzyme was carried out under the same conditions as for the first Alfi-Gel blue chromatography, except that the elution was performed with a 0-1 M NaCI gradient . The resultant active fraction IIbI was concentrated using polyethyleneglycol and dialyzed against 10 mM Tris-HCI, 10 mM CaCI Z, 0.75 M NaCI, pH 8.0. Three hundred microliters of the fraction IIbI was then applied to a column (0.9 x 45 cm) of Sephadex G-75 (superfine). The purified fibrinolytic enzyme (jararafibrase II) was obtained by collection of the fibrinolytic activity peak as shown in Fig. 5. The purification procedures are summarized in Table 1. The purity of the isolated enzymes, jararafibrase I and jararafibrase II, was confirmed by analytical PAGE. The isolated jararafibrase I and jararafibrase II revealed a single protein band on analytical PAGE. Jararafibrase I and jararafibrase II had a mol. wt of 47,000 t 2000 and 21,400 ± 500, respectively, by SDS-PAGE (mean t S.D . of six determinations) (Fig. 6). Under reducing conditions, both enzymes showed slightly increased mol. wts of 64,000 ± 1700 and 24,700 f 800, respectively (mean ± S.D . of three determinations) but no splitting of the molecules was observed. Isoelectric focusing was carried out in agarose gel . The isoelectric points ofjararafibrase I and jararafibrase II were 4.6 and 6.5, respectively . Jararafibrase I was strongly inhibited by ethylenediaminetetraacetate (EDTA), 1,10phenanthroline and dithiothreitol . However, no inhibition was observed with other serine

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