A thrombin-like enzyme from bushmaster (Lachesis muta stenophyrs) venom

0041-0IOl/93 56.00 + .00 ® 1993 Pmiamoo Pns Ltd

Tosieaw, Vd . 31, No. 11, pQ. 143~1443, 1993. p,inted ~ ~at ~.

A THROMBIN-LIKE ENZYME FROM BUSHMASTER (LACHESIS MUTA STENOPHYRS) VENOM F»EItIOO ARAGON-ORTIZ I and FRANC Gusi?tvv~Ex2 'Department of Biochemistry, Sehpol of Medicine, University of Costa Rica, San Jose, Costa Rica; and ~Depairiment of Biochemistry, Josef Stefan Institute, Jamova 39, SLO-61000, Ljubljana, Slovenia (Received 31 Mmch 1993; accepted 18 May 1993)

F. ARAGON-ORTrz and F. GuBEN~c. A thrombin-like enzyme from bushmaster (Lachesis mots stenophyrs) venom. Toxicon 31, 1431443, 1993 .-The clotting enzyme (Stenoxobin), from the venom of Lachesis mots stenophyrs, was purified by gel chromatography on Bio-gel P-100 followed by agmatine CH-Sepharose-4B and FPLC on Mono Q column . By SDS polyacrylamide gel electrophoresis the mol. wt was found to be 37,000 . The enzyme is a glycoprotein with 1 .6 moles of sialic acid per mole of protein and has an average content of 7 .0% of neutral carbohydrates. The clotting and esterolytic (BAEE) activities were 843 NIH units/mg and 60.1 f 1 .2 ODD ml/min/mg, respectively, and could not be inhibited by heparin or hirudin. Amino acid analysis revealed a low content of tryptophan and a high content of acid residues. Stenoxobin acts upon human fibrinogen by releasing consecutively fibrinopeptides A and B from the alpha- and beta-chains of fibrinogen. INTRODUCTION

Lachesls mots is a Central and South American Crotalidae snake, known in Costa Rica as cascabel much . It is found from Southern Nicaragua to Brazil . So far four different subspecies are known: L. m. mots, L. m. noctivaga, L. m. stenophyrs (VIAL and Ji1~NEZ-PORRA3, 1967) and L. m. melanocephala (SOLORZANO and CERDAS, 1986). In Costa Rica only the latter two subspecies are found. Lachesis mots stenophyrs inhabits the Atlantic and Southern Pacific zones of the country (TAYLOR, 1951). Its venom exhibits a significant clotting activity (ARAGON-ORTIZ, 1986), similar to other Crotalidae venoms . This activity is in part responsible for the defibrinogenating syndrome found in humans bitten by these snakes (BARRANThs et al., 1985). So far, thrombin-like enzymes from two subspecies L. m. noctivaga and L. m. mots have been described (MAGALHAES et al ., 1981 ; VII~IGAL SILVEiRA et al., 1989), but they are different from the enzyme found in the venom of L. m. stenophyrs . Owing to the clinical and biochemical importance of clotting enzymes, we isolated and further characterized this thrombin-like enzyme . MATERIALS AND METHODS The venom from Lachtsis mots sunoplryrs was obtained from the Instituto Clodomiro Picado, University of Costa Rica. It consisted of pooled lyophilized samples obtained from snakes captured in the Atlantic zone of the country. Substrates for esterase activity determinations, N-alpha-benzoyl-L-ar~nine ethyl ester (BAEE), N-alphaP-tosyl-L-lysine methyl ester (TLEME) and N-alpha P-tosyl-L-arginine methyl ester (TAME), agmatine 1435

1436

F. ARAGON-0RTIZ and F . GUBEN$EK

HCI, 1 ethyl-3-3(3-dimethyl-aminopropyl) carbodiimide HCl (EDC), guanidine HCI, heparin, Tris (hydroxymethyl) aminomethane and hirudin, were from Sigma Chemical Co . (St . Louis, MO, U .S .A.) . Molecular weight and iscelectric focusing protein standards and Sepharose 4 H were from Pharmmacia Fine Chemicals (Sweden) . Bio-gel P-100 was from Bio-Rad Laboratories (Richmond, CA, U .S .A .) . Human fibrinogen grade L, 90% clottable and the substrate 5-2238 were from AB-Kabi (Stockholm, Sweden). Topostasine was from F . Hoffmann La Roche A.G . (Basel, Switzerland). Protein test mixture 9 (pl-marker protein kit) was from Serve (Heidelberg, Germany) . Sodium chloride and sodium acetate were from Merck (Darmstadt, Germany) . Acetic acid was from Kemika (Zagreb, Croatia). FPLC system and gel electrophoresis apparatus GE-2/4 LS were from Pharmacia (Sweden). Analytical isoelectric focusing was done with a' Deaaga Deaaphor apparatus . Amino acid analyses were done on a Heckmann 118CL analyser . High performance liquid chromatography was done on a LDC Milton Roy apparatus . Polyacrylamide (7.5%) gel electrophoresis in Trite-glycine buffer (pH 8 .4) was performed according to the method of Days (1964) with a 4 .5% gel spacer. Polyacryhunide gel electrophoresis, in the presence of SDS, was made according to the method of L~r (1970) in 4-30% (w/v) polyacrylamide gradient gel . Before SDS electrophoresis, samples were heatod at 100°C for 5 min in Tris-HCI buffer (pH 6 .8) containing 1 % SDS and 5% 2-mercaptcethanol . Molecular weight markers were run in parallel on the same gel. Analytical iscelectric focusing was made on a l mm thick 5°/. polyacrylamide plate with carrier ampholines of pH 3-10. Protein test mixture 9, pl-marker kit, with known isoelectric points was run parallel on the same gel. All gels were stained for protein with 0.1 % Coomassie brilliant blue R-250 . Proteolytic activity was measured by the method of AwsoN (1939), using a 1 % solution of casein in O.l M phosphate buffer (pH 8 .0). The determination of the specific clotting activity was made according to the method of BeuatnruvN (1970) using human fibrinogen and bovine thrombin. The determination of the specific esterolytic activity was measured according to the method of Hl~r. (1959), using BAEE, TLEME and TAME as substrates. Inhibition by heparin was studied by incubating LOpg of the enzyme with different quantities of heparin ((0-50 heparin LU . per ml). The clotting time was then recorded . Inhibition by hirudin at 25°C was measured by following the hydrolysis of the chromogcnic substrate 5-2238 (o-Phe-pip-ArgpNa) at 405 nm . Different concentrations of hirudin (up to 1 .4 pM) were incubated for 10 min at 25°C with 20 to I00 pl (I00'rg/ml) of the enzyme . The mixture was then added to 100 frl of the substrate (2 mM), pH 8.1 . Sialic acid was determined in triplicate according to the thiobarbituric method of W~resex (1959). Neutral carbohydrates were determined according to the method of Wrrtzt.Ert (1961) in triplicate and expressed as galacto~e-mannose in la ratio. For the determination of the absorbance coefficient, the enzyme was exhaustively dialysed against distilled water, lyophilized and weighed on a microbahrnce . The weighed enzyme was dissolved in O .l M Tris-HC (pH 7 .3) and the optical density recorded at 280 nm in a 1 car cuvette. N-terminal amino acid was determined according to the double ooupliag method (Cttexa et al., 1978 ; Ar.r.itnt, 1981) . For amino acid analysis, samples were hydrolysed in duplicate with 4 M methane-sulphonic acid at 110°C in a nitrogen atmosphere for 22, 48 and 72 hr, rospectively. Amino acids which are subject to disintegration were interpolated to zero time value. Purific8tion of the clotting enzyme was performed in the following way: 500 mg of the venom was dissolved in 6 ml of the starting buffer (0 .1 M sodium acetate, 0 .1 M sodium chloride, pH 5 .5), centrifuged at 6000a and applied to a 2 .0 x 90 cm column packed with Bio-gel P-100. The column was equilibrated and eluted with the same buffer . The flow rate was 6 ml/mim and 2 .5 ml fractions were collected, read at 280 nm and tested for clotting of fibrinogen . The active fractions were pooled, concentrated by ultrafiltration with a UM-10 membrane and equilibrated with Tris buffer (0 .01 M, pH 8 .1) and applied to a column (1 .7 x 40 cm) packed with agmatine CH-Sepharose 4B (Arucorr-Oxrrz and Gtnretvlh, 1981) . The elution was performed with O.ISM NaCI in starting buffer, followed by 0 .15 M guanidine HCI in the same buffer. The fractions with the highest activity were pooled, concentrated and chromatographed by gel filtration under the conditions described above. The active material found in the second peak was rechromatographed under the same conditions, dialysed against 0.01 M Tris, pH 8.5, and applied to FPLC using a Mono Q column previously equilibrated with the same buffer . A gradient to 0 .25 M NaCI was applied over a time interval of 45 min in the same buffer . Clotting activity was pooled for further characterization. The release of fibrinopeptidea from human fibrinogen by stenoxobin and bovine thrombin was followed by high performance liquid chromatography (HPLC) with a Lichrosorp RP-18 analytical column . Human fibrinogen was dissolved in distilled water at a concentration of 8 mg/ml . Thrombin or the clotting enzyme stenoxobin, at a concentration of 10 NIH units, were added to 1 ml of fibrinogen which clotted within 60 sec. Each clot was incubated at 37°C at different time intervals. The reaction was stopped with 200 pl of 5'/o phosphoric acid and ûltered . The released peptides were determined in 100 pl aliquots applied to a HPLC column equilibrated with 10°/. acetonitrile in 0.1 % trifluoroacetic acid (TFA). After an initial wash with the starting solvent, the peptides were eluted by 50 min linear gradient to 50% acetonitrile at a flow rate of 1 .0 ml/min . Peptides were detected by absorbance at 220 nm and the peptide peaks, collected and dried in vacrro . The residues were hydrolysed with 6 M HCl at l 10°C for 24 hr and analysed for amino acid composition . RESULTS

The fractionation of the crude venom by gel filtration is shown in Fig. 1 . The clotting activity was found only in the first peak. After affinity chromatography on agmatine-

Thrombin-like Enzyme from Buchmaster

III N

V

VI

V SO

100

150

1437

\ 200

TUBE N0 . FYa . 1 . GEL 171LTf1A110N oN Blo-aEl, P-100 coLUCav (90 x 2 cm) of 500 mg a+ L. m. stenophyrs veivoa tN 0.1 M Na Ac., 0, l M NaCI pH 5.5, AT A FIAW aAlE OF 6 ml/min . All clotting active material was found in peak I ac indicated by bar .

Sepharose column the enzyme was further purified by FPLC (Fig. 2). High clotting activity was present in fractions I to VI which were pooled for characterization . Polyacrylamide gel electrophoresis of the purified enzyme and of the crude venom is shown in Fig. 3A. A single broad band corresponding to the enzyme is clearly seen . It is

Fta.

2. FAST r~tpoxaANCe uQtltn cEatoaArooaAt~tty of

rl~ cwrnxc

1~iZYaE oarA1N» NOLLOWINO AFFRYrI'Y CHROaATOCiRAPHY AND (lEI. FIL'rRA170N. Mono Q column (8 ml bod volume) . The enzyme waa eluted with NaCI gradient in 0 .01 M Tris-HCl at a Sow rate of 1 mlJmin . High clotting activity was found in peaks I to VI .

1438

F. ARAGON-ORTIZ and F. GUBEN$EK

mol. wt 94A00

pl

67,000 43,000

8.45 6.85

30,000

5.20

20,000

4.55

14,400 A

B

C

Ftc. 3. (A) Polyacrylamide 7.5% gel electrophoresis in Tris-glycine buffer at pH 8.4 of the pooled clotting enzyme after FPLC ; left lane : 150 hg of crude L. m. sttnophyrs venom; right lane : 40 Pg of pure stenozobin. (B) SDS gel electrophoresis in 4-30% (w/v) gradient gel of the clotting enzyme in the presence of 5% mercaptcethanol; left lane : 20pg of pure stenoxobin, right lane : mol. wt standards (lOpg each of the following proteins were applied: phosphorylase B, albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lactalbumin) . (C) Analytical iscelectric focusing on l mm thick 5% polyacrylamide plate with carrier ampholines of pH 3-10 ; left lane : standard proteins from the top: amyloglucosidase, soybean trypsin inhibitor*, beta-lactoglobulin A*, bovine carbonic anhydrase B, human carbonic anhydrase B, horse myoglobin-acidic band*, horse myoglobin-basic band, lentil lectin-acidic band, lentil lectin-middle band*, lentil lectin-basic band and trypsinogen (iscelectric points of bands marked with * are indicated); right lane : pure stenoxobin . The straight band at pl 4.55 is an artefact caused by the application of the sample.

located between the second and the third strong protein bands of the crude venom. SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol reveals a single polypeptide chain with mol. wt 37,000 (Fig. 3B). This estimated value was obtained by subtracting the carbohydrate moiety . The clotting enzyme is a glycoprotein containing 1.6 moles of N-acetylneuraminic acid per mole of protein and an average content of 7% of carbohydrates. The purified enzyme shows several bands in iscelectric focusing between pH 4.6 and 8.45, the strongest one being near pH 5.0 (Fig. 3C). Amino acid analysis and carbohydrate content of the enzyme is shown in Table 1 . The absorbance of 0.1% solution of the clotting enzyme stenoxobin in 0.1 M Tris-HCI, pH 7.3, was found to be 0.785 at 280 nm.

143 9

Thrombin-like Enzyme from Buchmaster TABI .E

1 . AlIINO

Amino acid Acz Thr Ser Glx Pro Gly Ala Cyc Val Met Ile Leu Tyr Phe His Lys Trp Arg

ACID CONP08I170N AND CON1EPrf OF SIF.AAOXOBIN

Found

CAABOHYDRA7E

(moles/mole)

Integer 34 16 18 31 I8 25 15 13 14 8 20 28 14 14 16 19 5 18

34.2 15.9 17.6 30.8 17.6 7A.5 15 .4 12.9 13.7 7 .9 19 .9 28 .3 13.7 14 .2 15 .9 19 .1 4 .9 17 .9

326

Total

Calculated mol. wt = 37,291 . The analysis was performed in duplicate . Standard deviation of the determinations is withiat4% . The protein contains on average 7% of neutral carbohydrates and L6 moles of n-acetylneuraminic acid per mole of protein.

The clotting activity of the crude venom was found to be 28.8 NIH units/mg and the purified enzyme had 843 IVIH units/mg. A 29-fold enrichment in clotting activity was obtained by purification . The enzyme also exhibits esterolytic activities hydrolysing arginine and lysine esters . BAEE is the most sensitive substrate (Table 2). Heparin did not inhibit the coagulation of citrated plasma by stenoxobin when present in a wncentration of 50 IU/ml, and 1 .4 ~tM hirudin did not inhibit its esterolytic activity toward 5-2238 . Characterization of the peptides released by stenoxobin was made by HPLC. The elution profile of peptides cleaved from human fibrinogen by the action of the clotting enzyme is shown in Fig. 4. The retention times of the first three peaks correspond to TeBIE

2.

SPECIFIC iS1EROLYTIC ACIiVITY OF L . vENOI~ ~ND rIS cco~rnNa ENZVe~

nl, stenophyrs

Substrate

Venom

Enzyme

BAEE TAME TLEME

0.769 f 0.012 0.246t0 .019 0.049 f 0.003

60 .1 t 1 .2 35 .8t 1 .0 1 .39 t 0 .07

Each experiment was done in triplicate with five different concentrations of protein . The average value of the respective activity was obtained by linear regression. The hydrolysis of BAEE was followed at 225 nm, TAME and TLEME at 247 nm . The activity (mean f S .D.) is expressed in OD.mI/min .mg.

1440

F . ARAGON-0RTIZ and F. GUHEN~`EK

0.5

a W S

0.4 0.3

~ 0 .2

5

20

60

120

TIME f min 1 FIa.

4,

TII~ couHSE of ~ ap~p " Q+ of FIHwNDFErrIDFS (srElvoxoBIN) .

A

AND

B

HY

rte

cLOrnNC ENZVe~

The insert shows a typical HPLC separation of released fibrinopeptides, following prolonged incubation with stenoxobin, on Lichrosorp column equilibrated with !0% acetonitrile in 0 .1°/. trifluoroacetic acid and eluted by a 50 min linear gradient to 50% acetonitrile .

fibrinopeptides A1, A2 and B. The elution profile of a mixture of supernatants from thrombin-induced and stenoxobin-induced clots shows that the first throe peaks overlap, only an increase in the height of the common peaks was observed. In Table 3, the amino acid compositions of the peptides released by stenoxobin are compared to the known amino acid compositions of fibrinopeptides A and B released by bovine thrombin . The peptides from the first two peaks (A1, A2) correspond to fibrinopeptide A, the third peak (B) corresponds to fibrinopeptide B (EBERT and B>~ .L, 1985). The time dependence of the release of fibrinopeptides A1, A2 and B from human fibrinogen by stenoxobin is shown in TABLE

Aax Ser Glx Gly Ala Val Leu Phe Arg

3.

A1SLN0

ACID COè~(~I170N OF RELEASED BY 31ENOXOHIN

FIBRINOPEPTIDFS

A,

Peaka Az

B

FPA

FPB

2 .19 1 .00 2 .32 5.19 2.06 0.60 0.98 0.91 0.57

1 .80 1 .00 1 .98 5 .27 1 .59 0 .90 1 .00 1 .05 0 .88

2 .65 L00 2 .65 2 .I1 1 .03 0 .82 1 .59 0.50

2 1 2 5 2 I l 1 1

3 l 3 2 1 1 2 I

The analysis was performed in duplicate . Results are given in moles. Standard deviation of the determinations is within f 3% .

Thrombin-Gke Enzyme from Bushmaster

144 1

Fig. 4. It can be seen that during the first 5 min of incubation of the clot at 37°C, moat of the fibrinopeptide A is released . Fibrinopeptide B is released at a slower rate, approaching the maximum value only after 8 hr of incubation. The N-terminal sequences of fibrinopeptides A1 and A2 correspond to the known sequence of the peptide A. Fibrinopeptide A1, designated as AP by BL.o~Acx et al. (196, has a phosphorylated Ser in third position. For the peptide present in the third peak (B), no PTH derivative was obtained; however, its amino acid composition corresponds to fibrinopeptide B. It is known that fibrinopeptide B from human fibrinogen has a pyroglutamic acid residue at the N-terminus and thus, no direct derivative could be obtained .

DISCUSSION

The clotting enzyme stenoxobin was isolated from the venom of Lachesis muta stenophyrs. The physicochemical properties of stenoxobin are similar to those of clotting enzymes isolated from the venoms of other snakes such as Agkistrodon rhodostoma (ESNOUF and TUNNAH, 1967), Bothrops moojeni (STOCxE~t and Bwitr..ow, 1976), Crotalus adamanteus (MwxxLwxn and DwIKUS, 1971), Agkistrodon acutus (OUYANG and YANG, 1974), Bothrops riper (AxwcoN-ORTIZ and GvaEx~c, 1981) and Trimeresurus flavoviridis (Si-m?t-t et al., 1985). These clotting enzymes are glycoproteins, with a single polypeptide chain and mol. wt around 35,000, and in addition, they exhibit esterolytic activities which are not inhibited by heparin. Stenoxobin also shares some of the properties of viperabin, the thrombin-like enzyme from the venom of Cerastes vipera, the Sahara sand viper (ELAs~wlt et al., 1992). In contrast to thrombin, the esterolytic activity (measured toward the S-2238 substrate) of the L. m. stenophyrs clotting enzyme is not inhibited by hirudin. Comparing the properties of stenoxobin with the clotting enzymes from L. m. noctivaga (MAGAi .HARC et al., 1981) and from L. m. muta (VII)IGAL S>t.vEaew et al., 1989) the following observations can be made : stenoxobin appears to be heterogeneous as judged by bands of different pi values and the FPLC pattern of purified enzyme . This heterogeneity is attributed to different levels of syalidation of the enzyme, as well as to the differences in charged polysaccharide moiety . In contrast, only a unique pl value of 5.1 is reported for the L. m. noctivaga enzyme. Heterogeneity was also found is the L. m. muta enzyme whose iscelectric points are between 3.1 and 5.0 . The specific clotting activity of stenoxobin is only 53% of Lachesis muta noctivaga enzyme and 70% of L. m. muta enzyme. Its mol. wt (37,000) and amino acid composition are quite similar to the L. m. noctivaga clotting enzyme (36,300), but it differs significantly in these properties to the clotting enzyme from L. m. muta (mol . wt 41,000 to 47,000). Stenoxobin releases fibrinopeptides A and B from the alpha- and beta~hains of human fibrinogen . Fibrinopeptide B is, however, released at a lower rate than fibrinopeptide A. In this action stenoxobin is similar to thrombin (BL.ot~cx et al., 1978; MARTI1dSL~-Ir and Si~twcw, 1980) and differs from L. m. muta enzyme and other thrombin-like enzymes from snake venoms that only release fibrinopeptide A (MwRxi.wND and DAMUS, 1971 ; STOCKER and BARLOW, 1976). Some exceptions are known: the enzyme from Agkistrodon contortrix contortrix, which releases fibrinopeptide B first and then fibrinopeptide A (Hiatzlc et al ., 1970), and the enzyme from the gaboon viper Bibs gabonica which cleaves both fibrinopeptides A and B (MAR,SH and WIIwL>~t, 1974 ; PIItxL.E et al., 1986). The clotting enzyme stenoxobin did not show proteolytic activity toward casein when incubated at an enzyme to substrate ratio of 1:50 for 3 hr at 37°C .

1442

F. ARAGON-0RTIZ and F. GUBEN$EK

Since the clotting enzyme from Lachesis mute stenophyrs releases fibrinopeptides A and B during the coagulation of fibrinogen and also hydrolyses Arg and Lys esters, we may conclude that stenozobin is a true thrombin-like enzyme of snake venom origin . Atkrwwledgemcnts-This work was supported by the Ministry of Science and Technology of Republic of Slovenia . F®attoo Aiucoty-Oxrtz is a research fellow of the Costarican National Council for Science and Technology (CONICIT), contract 89727475 J. REFERENCES Ar tErr, G. (1981) Sequencing ojProteins acrd Peptides. Amsterdam: Elsevier. Atrsotv, M. L. (1939) Estimation of pepsin, trypsin, pepsin and cathepain with hemoglobin . J. gen. Physiol. Z2, 79-59. Axncort-0trnz, F. (1986) Purification and properties of a coagulant proteinase isolated from bushmaster (Lachesis trams) venom. Rev. Blot. Trop. 34, 55-58. Atuaox-OxTtz, F. and GuaEtv~, F. (1981) Bothrops riper venom of Atlantic and Pacific zones of Costa Rica. Toxicon 19, 79705. Bexxerrtrs, A., Sot.ts, V. and Horwxos, R. (1985) Alteration de los mecanismos de la coagulation en et envenenamiento por Bothrops riper (terciopelo) . Toxicon 23, 399-~07. Beuo~nurnv, D. J. (1970) Thrombin assay. Meth . Enzym. 19, 145-157. Bt.o~xcc, B., Htamxcrt, M., Ent~tv, P. and Ht~., B. (1966) Human fibrinopcptides. Isolation, characteri~tion and structure. Blochine . blophys. Acts 115, 371-396. Bt .o~mlcrt, H., Ht~., H., Hoax, D. and Tt~ecn~serr, L. (1978) A two-step fibrinogen-fibrin transition in blood coagulation. Nattoe 275, 501-505. C~tntvo, J. Y., Bw~uett, D. and Wrrreux-L®ot.n, B. (1978) Micro sequence analysis of peptides and proteins using 4N.N-dimethylaminoazobenzene 4'-isothiotyanate/phenylisothiotyanate double coupling method. FEBS Lett. 93, 205-214. Dwrs, B. J. (1964) Diet electrophoresis. 2. Method and application to human serum proteins . Ann. N. Y. Aced. Sci. 121, 404-427. EeEaer, R. F. and Bets ., W. R. (1985) Assay of human fibrinopeptide by high performance liquid chromatography. Analyt . Biochem . 148, 7O-7ß. Et.-Asewe, M. F., Fnxm, T. M. and Nest, H. (1992) Purification and partial characterization of viperabin a thrombin-like enzyme from the venom of Cerastes vipers (Sahara sand viper). Toxicon 30, 504-505. EsNOUt", M. P. and Turttaex, G. W. (1967) The isolation and properties of the thrombin-like activity from Agkistrodon rhodostorna venom. Br. J. Haemat . 13, 581-590. Heteztc, R. H., Rerxot~, O. D. and Stiettvot~, J. R. (1970) Studies on a coagulant fraction of Southern copperhead snake venom: the preferential release of fibrinopeptide B. J. Lab. clan Med. 76, 45165. Hues., B. C. W. (1959) A modified apectrophotometric determination of chymotrypsin, trypsin and thrombin . Can. J. Biochem. Physiol. 37, 1393-1399. Leee~.t, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 68085. Mecetxe~, A., De Or .tvmtw, G. T. and Dtxtz, C. R. (1981) Purification and partial characterization of a thrombin-like enzyme from the venom of the bushmaster snake Lachesis mute rroctivaga. Toxicon 19, 279-294. Meau .etvn, F. S. and Dews, P. S. (1971) Purification and properties of a thrombin-like enzyme from the venom of Crorahrs admnartteus (Eastern diamondback rattlesnake) . J. biol. Chem. 246, 6460-6473. Mexstt, N. A. and WnetEa, B. C. (1974) Separation and partial characterizadon of a coagulant enzyme from Bibs gabonica venom. Br . J. H~mat. 26, 295-306. Mwant~.tr, R. A. and Sc~reae, H. A. (1980) Steady-state kinetics study of the bovine thrombin-fibrinogen interaction. BiochemLttry 19, 2343-2350. Ouverra, C. and Yexa, F. Y. (1974) . Purification and properties of the thrombin-like enzyme from Trimeresrrrrrs gramineua venom. Biochim. blophys. Acts 351, 354-363. PlttxtE, H., Trmooox, L, MtYene, D, and Shorts, D. (1986) Thrombin-like enzyme from the venom of Bitis gabonica. J. bfol. Chem . 261, 8830835. Stt>Bt, T. C., TeMeee, S., K1FteAe, H., Ottrto, M. and Metasuua, S. (1985) Purification and characterization of a coagulant enzyme of Trhneresurus flavoviridis venom. J. Biochem. 9B, 7I3-721 . Sot.oxzerto, A. and Cracow, L. (1986) A new subspecies of the bushmaster, lachesis mute from Southeastern Costa Rica. J. Herpet . 20, 463~t65. Sroctmt, K. F. and Betet.ow, G. H. (1976) The coagulant enzyme from Bothrops atrox venom. In : Methods in Enzymology, pp . 214223, Vol. XLV (Cowwtctr, S. P. and IüPt.ert, N. O., Eds). New York: Academic Press. TeYt.on, E. H. (1951) A brief review of the snakes of Costa Rica . Univ. Kansas Sci. Bull. 34, 3-188.

Thrombin-like Enzyme from Buchmaster

1443

Vus ., 1 . L. and Jn~IBZJ. M . (1%7) The eco®eography of the buchmatter, Lachesis muta, in Central America. Am . Midi. Natty . 78, 182-184. VmtaU. Su vt~, A. M ., MAa~r ra~na, A  Dnvtz, C. R . and Dt: OLIV~tA, E. B. (1989) 1?uriücation and properties of the thrombin-like enzyme from the venom of l achesis muta conta. let. J. Blodean. 21, 863-871 . W~we~t, L. (1959) The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975 . wINZIF1t, R. J. (1%1) Determination of serum glycoproteins. In: Methods of Biochemical Aealystr, pp . 29t?-292 (Guc~, D., Ed .) . New York : Interscience .