Toxtcon, Vol. 17, pp. 145-134. O Peraamoa Pmn Ltd. 1979 . Printed is l3re~t HrltYin.

0041-0101/79/0301-014350200/0

SEPARATION OF BITIS GA BONICA (GABOON ADDER) VENOM ARGININE ESTERASES INTO KININ-RELEASING, CLOTTING AND FIBRINOLYTIC FACTORS CORNELIS C. VII,IOEN, CARMEL M. MEEHAN and DAWIE P. BOTES National Chemical Research Laboratory, Council for Scientific and industrial Research, P.O . Box 395, Pretoria, 0001, South Africa

(Accepted for publication 17 July 1978) C. C. Vn.loax, C. M. MasaAN and D. P. Bores . Separation of Bitas gabonica (Gaboon adder) venom arginine esterases into kinin-releasing, clotting and fibrinolytic factors. Toxicon 17,145154, 1979. The alginine ester hydrolysing activity of the venom of Bibs gabonica has been isolated and purified by a combination of Sephadex gel filtration and ion-exchange chromatography on DEAE-0ellulose. The esterases were designated E-I, E-II and E-III according to their sequence of elution from DEAE~ellulose . A molecular weight of about 32,000 was calculated for the enzymes which contain 20-25 ~ by weight of carbohydrate . The enzymes appear to be specific for arginine esters, since lysine esters are not hydrolysed, and showed low proteolytic activity against casein . Distinct biological properties were found for the estelases, E-I, EIl and E-III showing kinin-releasing, fibrinolytic and clotting activity, respectively . VENO~IS from most

INTRODUCTION

species in the families Crotalidae and Viperidae are known to be particularly rich in a wide spectrum of hydrolases, including several types of proteinases. A further distinction into fractions showing proteolytic activity towards casein and arginine esterases have been shown (TU, 1977 ; OSHIMA et al., 1969 ; HENR1QuES and EVSEEVA, 1969). MESS (1970x) in a comparative study of enzyme activities in snake venoms, found Bitis gabonica to contain both activities and which, by using Sephadex G-SO gel filtration, were subsequently resolved into two separate fractions by BOTES and VIIJOEN (1974) . The present report is concerned with the further resolution of the esterase activity into various enzyme fractions all of which are active toward arginine esters but are distinguished by diverse biological functions. MATERIALS AND METHODS Lyophilized Bibs gabonica venom was purchased from John Visser, P.O . Box 20, Camps Bay, South Africa. Sephadex (Pharmacia) and Whatman DEAF-cellulose, DE-52 (W . & R. Ballton, England), were prepared for column chromatography as recommended by the manufacturers . N-a-tosyl-L-arginino-methyl ester (TAME), N-0,-acetyl-L-tyrosine ethyl ester (ATEE), N-a.-benzoyl-nL-argininel3-nitroanilide (nl.BAPA) and N~.-benzoyl-nrrlysine~l-nitroanilide (nrrBLPA) were purchased from Merck A. G. (Germany) Nß-benzoyl-rrargine ethyl ester (BAEE) and molecular weight markers for SDS polyacrylamide gel electrophoresis were obtained from BDH Laboratories (England). Bradykinin. and Nit.-acetyl-z_lysine methyl ester (ALMS) were supplied by Sigma (U .S .A .) and bovine thrombin and bovine fibrinogen, fraction I, 90 ~ clottable, by Nutritional Biochemicals Corporation (U.S.A .) and Miles Laboratories (U.S .A .) respectively . Trypsin (2 x crystallized, salt free, diphenylcarbamyl chloride treated) was bought from Seravac Laboratories, Cape Town, Republic of South Africa, chymotrypsin (3 x crystallized) from Worthington (ü.S :A.) and neuraminidase from P-L Biochemicals (U.S .A .).

Electrophoresis and molecular weight determination

Disc elechophoresis on 7~5 ~ polyacrylamide gels at pH 8~1 was performed according to the method of Rsrsr>a n and St~u. (19C~ and SDS polyacrylamide gel electrophoresis was done essentially as described 145

146

CORNELIS C. VILJOEN, CARMEL M. MEEHAN and DAWIE P. BOTES

by WEHER and OSHORN (1969) as modified by ALSREQiT and VaN Zvt. (1973) . The molecular weights of purified enzyme fractions were deternvned by SDS gel electrophoresis using molecular weight markers in the range 14,300-71,500 . The markers and proteins were prepared for electrophoresis as recommended by the manufacturers . Molecular size was also estimated by gel chromatography of the reduced proteins in 6 M guanidine hydrochloride as described by Ftsx et al . (1969) . The columns were calibrated with standard proteins (FtsH et a1 .,1970) andthedata were plotted aaording to the suggestion of ActcERS (1967) . Molecular weights were obtained from a logarithmic plot of Stokes radius (R,) vs molecular weight for standard proteins . Amino acid analysis Protein samples were hydrolysed in redistilled constant boiling HCI, containing 02 ~ (w/v) phenol in evacuated, sealed tubes. Amino acid analyses were performed on the automated Beckman 120B amino acid analyser, while tryptophan and tyrosine were calculated spectrophotometrically according to the method Of GOODWIN and MUR7UN (1946) . Protein concentration Protein concentration was calculated according to the method of SCOPES (1974), using absorbante values at 205 and 280 nm . This estimate of concentration was used in conjunction with absorbance values at 280 nm to derive Ate'°t values . Carbohydrate analyses

Carbohydrate content was deternvned according to the following methods as described by WINZLER (1955) : protein bound hexoses by the oroinol-H,SO, reagent, hexosamines by the Elson-Morgan method, methyl pentose with the aid of the H,SO,i:ystéine reagent and sialic acid by the tryptophan-perhloric acid reagent. Enzyme assays Proteolytic activity was assayed accordingto the method of Kurerz (1947),as described by Mass (1970a), using 1 ~ casein solution in 0"1 M Tris-HCI buffer, pH 7-8. One unit of enzyme activity was defined as the amount of enzyme which caused an increase in Ao of 0001 unit per min. Arginine ester hydrolysis was determined by the method of SCHWERr and TARENAKA (1955) using HAEE as substrate. Enzyme solution was added to 2~5 ml of substrate solution in a 1 tm cuvette (0 "5 mM BAEE in 01 M Tris-HCl buffer, pH 7~8) and the increase in absorbency at 253 nm recorded . One unit of enzyme activity was defined as the amount of enzyme which caused in 1 min an increase in absorbency of 0001 . Esterolytic activity against TAME was assayed identically, but using the increase at 247 nm as basis for calculations . Chymotrypsin type activity was measured according to the method of SCxwERT and TAItENAYA (1955) using 1 mM ATEE in 0" 1 M Tris-HCI, pH 7-8 containing 0"1 M CaCI,, as substrate by recording the decrease in absorbante at 237 nm . Trypsin-like peptidase activity was determined by the method of ERIdNOER et al. (1961) using as substrate 1 mM Dt-BAPA and 1 mM-BLPA in 0"1 M Tris-HCI, pH 7"8 containing 0" 1 M CaCI,, and recording the absorbents increase at 410 nm wntinuously. One unit of activity was defined as the amount of enzyme causing an increase of 0001 absorbency unit in 1 min . Fibrinolysis was measured as described by KuNE (1955) . To 1 ml of fibrinogen solution (0"5 ~ in 01 M borate buffer, pH 7"75), 1 ml of test enzyme solution (containing ~ 100 kg of enzyme in 0"1 M borate buffer pH 7"75) and 0~2 ml of thrombin (20 units/ml) were added. The contents of the tubes were mixed, incubated at 37°C and the lysis time for complete disappearance of the fibrin clot noted. Coagulase activity was determined as set out by DEVt et al . (1972) using 06 ~ fibrinogen M sodium phosphate buffer, pH 7~0, containing 0~1 M NaCI . Bovine thrombin (180 units per mg) was used to construct a standard curve of clotting time against thrombin units. Samples were incubated at 3? °C. Kinin-releasing activity was measured according to the description of MEHS (1970a), using synthetic bradykinin as standard . Enzyme solution (005 ml) was incubated for 3 min at 37°C with 3 ~ bovine globulin (plasma fraction precipitated betwcen 03 and 0"5 fold ammonium sulphate saturation) in Tyrode solution . An aliquot of the solution was tested on isolated guinea pig ileum (in l0 ml aerated Tyrode solution with 1 mg atropine and 0~1 mg Avil per litre, at 37°C). The amount of liberated kinin was calculated from the rate of ileum contraction and one unit of activity defined as the amount of enzyme which released kinin equivalent to 1 pg of bradykinin per nvn. RESULTS

Figure 1 shows the separation obtained when 100 mg of the Sephadex fraction containing BASE hydrolysing activity (see Fig. 1, BOTE4 and Vu..TOErt, 1974) was applied to a DEAE-cellulose column at 5°C. Rechromatography of the esterolytic fractions E-I, E-II and E-III on DEAE-cellulose under similar conditions as those described in Fig. 1, resulted in esterase fractions E-I, E-II and E-III (Fig. 2) . This material migrated as single

147

B. gabonieu Ar~inine Fsterases

gads on both ordinary and SDS polyacrylamide gel electrophoresis (Fig. 3) and appeared to be uncontaminated by other components . Judged by their electrophoresic mobilities on SDS polyacrylamide gel electrophoresis, the esterases have a molecular weight ofca. 29,000 . This value was conßrmed by the results from gel chromatography in 6 M guanidine hydra chloride where an estimate of 32,000 was obtained (Fig. 4). The Stokes radii (R,) of the particles under these circumstances were found to be 54 A as compared to an R, of26 "8 A measured by a calibrated Sephadex G-75 column in the absence of denaturing agents. The minimum possible radii of the particles (R~, corresponding to the radius of a perfect sphere, was calculated as 20"8 A using 32,000 as molecular weight and equation (10) of Texroxn et al. (1974) . The enzymes are very similar in amino acid content (Table 1). E-III appears to differ from E-I and E-II only with regard to the tryptophan content. Table 1 also illustrates the values used for the determination of protein concentration from absorbances at 280 nm. The esterases contain a rather high amount of carbohydrate (20-25~, Table 2). Electrophoretic heterogeneity between E-I and E-II appears to be related to their sialic acid content, since they became electrophoretically identical upon incubation with neuraminidase (Fig. 3). Digestion was carried out in 0"1 M ammonium acetate, pH 5"5, containing 0"1~ CaCI, and 1 ~ NaCI to which neuraminidase was added at an enzyme to esterase ratio of 1 :20. Incubation was continued for 40 hr at 37°C. Although the digestion did not affect the esterase activities of fractions E-I-E-III, it caused E-I and E-II, to move together and changed the electrophoresic conduct of E-III, as shown in Fig. 3. However, a charge difference between the latter and E-I and E-II is still evident. o~a o' e o'a 0 M N

a

03 m

F 2

0'2

W

a

Y N i W

0'I

~,~ i'1V .

1.

~i . OF GRADIENT C:~ftOYATOORAPHY OF FRACIION II (~(i . B078s Al~ CELLIIIA~ .

1,

Vu .losx, 1974)

ON

DEAE-

tine hundred milli~alns of fraction II was dissolved and die(ysed against 0"001 M 1'rir-HCl, pH 7" 8 and subsequently applied to a DEAF-cellulose column (50 x 0" 9 cm) . Elution of the absorbed material was eûected by a 2 litre linear aladient of 0"001 M-0 "OS M 1~is-HCI, pH 7"8 . ( to~raphy was carried out at 3°C and arginine osberase activity determined as described in the test . The shadov~ed atea4 indicate material with esta~aso activity .

148

CORNELIS C. VILJOEN, CARMEL M. MEEHAN and DAWIE P. BOTES

0

F_ Z W

rN Z W

20 Î FIO . 2 . RECxROMATOGRAPkIY

40

30 OF

50

60

GRADIENT

FRACTIONS E-I, E-II AND E-III (FIG . 1) oN DEAE-ceu.uws~. Two hundred and ßfty milligrams of the particular fraction was applied to DEAE~ellulose (SO x 0~9 cm) at 5°C. Absorbed material was eluted by using a 4 litre linear salt gradient of 0001 M-0~OS M Tris-HCI, pH 7~8 . Shadowed areas represent material with esterase activity . of

I a

Is

l' 2

LD l

3'2

l

3 6

o

n

4~0

44

LOG MOLECULAR

WEIGHT

FYO . 4. LOOARTTHI~TIC PLOT OF Tl~ STORES RADIUS (R,) VERSUS MOLECULAR WEIGHT. A Sephadex G-200 column (80 x 1~5 cm) in 6 M guanidine-hydrochloride was calibrated with standard proteins : Chymotrypsinogen (In, cytochrome C (III), oxidized insulin B chain I represents the experimental point obtained forcaresses (IV) and oxidized insulin A chain E-I,. E-II and E-III. The esterases and chymotrypsinogen were initially roduced and elkylated as described (BoTES and VIIJOEN, 1974). Data were plotted according to the suggestion of AcxEIes (1%7}to derivaa value of R, which was >aubsequently used is tho-above relationship .

M.

FIG . 3 . POLYACRYLAMIDE GEL ELECTROPHORESIS OF E~TERASES E-I, E-II AND E-III . Electrophoresis on 40 ug protein was carried out at pH 8~1 for 4 hr with a current of 3 mA per gel . The gels were stained for protein using Coomassie Brilliant Blue . A contained esterase E-1, B esterase E-I I and C esterase E-III . A mixture of E-1, E-li and E-Ill was applied to D after digestion with neuraminidase as described in the text .

B. gobonico

Arginine EsteTases

Testa 1 . AMINO ACID COMPOS1170N E-I (Residues per mok) 26-27 14 14-15 18 18-20 17-18 16 9-10 10-11 7 14-1 S 19-20 T 8-9 16 8 6 6' 1 "37

Amino acid Asp Thr Ser Glu Pro Gly Als ~Gj~s Val Met Ire Leu Tyr Phe Lys His Arg Trp

p~mslm~

OF

15 1

FRACTIONS E-I, E-II ANn E-III

E-II (Residues per mole) 26-27 13 14 18 1&-20 18 16-17 8 10-11 7 1S 19-20 7" 8 16 8 6-7 6' 1 ß7

E-III (Residues per mole) 2027 14 15 19 18-20 18 16 9-10 11 6-7 15 19-20 7" 8-9 16 8 6-7 S" 1 ~ZO

"Determined spoctrophotometrically by the method of GoonwrN and MORTON (1946) . Tyr was determined by this method since it could not be evaluated from elution diagrams as hexosamines CO-elute with Tyr. TAwr-F 2 . CARBOHYDRATE OONTEM' OF FBTERA.4E FRACTIONS E-I, E-II AND E-III Fraction E-I E-lI E-lII

Hexose 9~7-11 " 1 9"7-11 " 1 6" 8-8~6

Sugar ( ~ by weight) Hexosamiue Methyl pentose 6"2-6 " 7 4" 1~ " 3 5"8~ " 0 4"2-4 "4 S " 1-S " 3 3"8

Sialic acid 3"7 4"3 3 "6

Table 3 sTlmr+~+ari~P s the results of experiments to characterize the enzyme activities of the purified fractions. All fractions were active against the arginine esters BAEE and TAME, and BAEE appeared to be a better substrate for the enzymes than TAME. Optimal esterase activity of the fractions against BAEE was observed at pH 8"5 (Fig. 5). The enzymes were inactive against ATEE and the amidase activity toward DL-BAPA was only about 5-6% that of trypsin . When nL-BLPA was substituted for Dl.-BAPA, no reaction was detected. The caseinolytic activities of the esterases were only about ~10~ ofthe activity oftrypsin . TAStE 3 . ENZYME ACTMTIE4 OF FRACTIONS E-I, E-II Arm E-IIIt

Fraction E-I E-II E-III

Activity against casein (units per mg) 53S 487 203

Activity against DL-BAPA (units per mg) 47 45 38

Activity against BAEE (units per mg) 5156 3449 3750

Activity against TAME (units per mg) 1016 1013 583

Kinin releasing activity (ugBr. per min per mg) 17 2~6 -

Clotting activity (Thrombin units per mg) 0" 03

Fibrinolytic activity -F +' -

" Positive Traction observed . tThe fractions had no activity against nT .-BLPA, ATEE and ALMS. The following abbreviations aTr used : nTrBAPA-N~.-benzoyl-nr.-argiaine~-nitroanilide ; nIrBLPAN-a-benzoyl-ntrlysine-4-nitroanilide ; ATEE-N-a-acetyl-rrtyrosine ethyl ester ; ALMIrN-a.-acetyl-Llysine methyl ester ; .BAEE-N~-benzoyl-t.-arginine ethyl ester ; TAME-N-0,-tosyl-rrarginine methyl ester.

15 2

CORNELIS C . VILJOEN, CARMEL M . MEEHAN and DAWIE P . BOTES

Three distinct biological activities could be ascribed to the enzyme fractions. Only E-III showed clotting activity when the purified enzymes were added to fibrinogen solutions (the enzyme contained an equivalent of f 003 thrombin units per mg) . Kinin-releasing activity was mainly associated with E-I on the basis of assays against bovine plasma, whereas E-II gradually lysed a clot produced by the action ofthrombin on fibrinogen.

ao P E

ÔO

~c ~E ô E c

40

M O

20

0

s,o

z,o

a,o

s,o

io,o

pH

FYo . 5 . Vexte~nox of xEncrtoty vELOCrrr wrt~ pH . Activities were monitored in a pH slat assembly, the reaction vessel being thecmoatated at 25° C . Reaction solutions consisted of 25 ml 5 x 10 - ' M BASE in 015 M KCl and the add liberated automatically titrated with 0002 N NaOH . The reaction vessel was continuously Bushed with nitrogen . DISCUSSION

A preliminary fractionation of B. gabonica venom on Sephadex G-50 (Bores and Vluoarr, 1974) indicated that the caseinolytic and esterolytic activities were probably associated with different enzymes. The arginine ester hydrolysing activity of the venom is shown here to be separable into fractions having kinin-releasing, fibrinolytic and clotting activity by Sephadex gel chromatography followed by ion exchange chromatography on DEAE-cellulose . A kinin-releasing enzyme, which might correspond to the enzyme E-I described in the present report, was also isolated by Mass (1970b) from Baboon adder venom by Sephadex gel filtration and chromatography on DEAE-cellulose and hydroxylapatite . The elution characteristics of esteraes E-I-E-III on DEAE-cellulose indicate an iso-electric point in the region of pH 7~0, which is probably the reason why Mebs could not separate the arginine hydrolyases on DEAF-cellulose. The material eluted by Mebs from hydroxylapatite with 001 M phosphate, pH 6~8 (Fig. 3, Mass, 1970b) was not further characterized, although Mebs noted that the fraction possessed high BAEE-hydrolysing activity. Possibly this fraction contained the esterases designated here as E-II and E=III. The esterases were previously found to be slightly retarded on Sephadex G-50 (Bow and VILJOEN, 1974), and on this basis a molecular weight of about 32,000 was assumed. The same value was calculated for the enzymes by gel exclusion chromatography and from the amino acid and carbohydrate content . The figure of 32,000 falls within the range of molecular weights reported for arginine esterases from other sources : 30,000 for the esterase from Agkistrodon contortrix laticinctus (Toots et al., 1970), Vipers ammodytes arnmodytes kininogenase, 29,500 (BAILEY and SI-i>POLIrtt, 1976) ; kallikreins A and B from porcine

B. goboxlca Arginine Estetases

15 3

pancreas, 25,300 (FIEDLER et al., 1975); rat urinary kallikrein, 32,000 (PORCF.LLI et al., 1975) ; batroxobin from Bothrops riper and B. moojeni, 32,000 and 36,000 respectively (STOCKER and BAItLOW, 1976); and crotalase from Crotalus adamautttts, 32,700 (MARKLAND, JR., 197 . Comparison of the Stokes radius derived by gel chromatography (R, = 26 .8 ~), with the minimum value (Rm,n) for a perfectly spherical unsolvated particle, indicated that the esterases behave as globular particles, R,JRm,o = 1 ~29 (VISSER et al., 1975). The enzymes contain large amounts of carbohydrate : our value of 20-25 is in good agreement with the values found for esterolytic enzymes from other venom sources. HATTON (1973) reported 29~ by weight ofcarbohydrate in the coagulant enzyme from A, rhodostoma venom, whereas BAILEY and SHIPOLINI (1976) reported that the kininogenase of V. ammodytes ammodytes contained 18-20 carbohydrate . Investigation of the enzymatic activities of fractions E-I - E-III established a pattern of activities similar to that observed for arginine ester hydrolyses from other snake venoms, e.g. the enzymes showed low proteolytic activity towards casein and showed an apparent specificity for arginine esters as lysine esters were not hydrolysed (compare Toots et al., 1970 ; Sox and CHAN, 1974 ; BAILEY and SxrnoL1NI, 1976). The esterases E-I-E-III therefore resemble trypsin as regards substrate specificity, but unlike trypsin, the enzymes were not inhibited by the potent trypsin inhibitor, ovomucoid . The alkaline pH optimum ofE-I-E-II, also agrees with previous observations made with esterolytic enzymes (Toots et al., 1970) BAILEY and SüIPOLINI, 1976 ; MARICLAND, JR ., 1976 ; SILVA et al., 1974) . However, the pH optimum of 8~5 (Fig. 5) is contrary to the value of 9~5 reported by MEHS (1970b) for the kininogenase he isolated from gaboon adder venom . The almost identical amino acid composition of esterases E-I-E-III suggests that the enzymes agree in primary structure. However, the enzymes were distinctly different with regard to biological properties which implies di$'erences in their active sites. The catalytic characteristics of the esterases are presently being studied and will be reported on in subsequent communications . It is already evident that within this group of esterolytic enzymes divergent functions have evolved which contribute to the prey-immobilizing and pain-producing defensive action of the venom . Very likely, as further information accumulates, the functional significance ofmost, ifnot all ofthe enzymes present in snake venoms will become apparent . Acknowledgements-We thank Dr. L. VtssEx of this laboratory for assistance in determining molecular weights by gel chromatography and Mr. R. Btwoto and Prof. J. J. Tm=ROx, Department of Physiology, Basic Medical Sciences, University of Pretoria for the kinin-releasing assays . REFERENCFS

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CORNELIS C. VILJOEN, CARMEL M . MEEHAN and DAWIE P . BOTES

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