Comp. Biochem. Physiol. Vol. 70B, pp. 349 to 351, 1981 0305-0491/81/100349-03502.00/0 Printed in Great Britain. All rights reserved Copyright © 1981 Pergamon Press Ltd

COMPARISON OF FIBRINOGENASES ISOLATED FROM COPPERHEAD VENOMS

JEFFREY B. MORAN and COLLIS R. GEREN* Department of Chemistry, University of Arkansas, Fayetteville, AR 72701, U.S.A.

(Received 25 February 1981)

Abstract--l. Fibrinogenase activity from fractions obtained by ion-exchange chromatography of whole copperhead (Agkistrodon contortrix) venoms were further purified by molecular sieve chromatography.

2. All four enzymes had apparent mol. wt of 23,000-24,000, both non-reduced and reduced as deter- mined by sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography.

3. All four enzymes rapidly degraded the ~-chain of the fibrinogen while apparently leaving the fl- and 7-chains intact.

INTRODUCTION

In the last several years a number of proteinases have been isolated from snake venoms which prevent the normal clotting of fibrinogen by enzymatically degrading the ~- or fl-chains (Ouyang & Teng, 1976; Ouyang & Huang, 1976, 1979; Ouyang et al., 1978, 1979). In earlier reports from this laboratory, Moran and associates (Moran & Geren, 1979a,b, 1980; Moran et al., 1979) noted the presence in copperhead (Agkistrodon contortrix) venom of both a thrombin- like (pro-coagulant) activity and a fraction which acted on fibrinogen to prevent clot formation. Recently Moran & Geren (1981) reported on the puri- fication and characterization of the fibrinogenase from the venom of the northern copperhead (A.c. mokasen) and preliminary work indicated that the fibrinogenase-containing fractions from the venom of the other four subspecies of copperhead had similar characteristics. However, because of subspecific differ- ences in phospholipases (phosphatide acyl hydrolase, EC 3.1.1.4) isolated from copperhead venoms (Moran et al., 1980), we decided to purify and partially charac- terize the fibrinogenases from the venoms of each of the other four copperhead subspecies.

MATERIALS AND METHODS

Lyophilized whole southern (A.c. contortrix), broad- banded (A.c. laticinctus) and trans-pecos (A.c. pictigaster) copperhead venoms were purchased from both Sigma Chemical Co., St. Louis, MO, and the Miami Serpentar- ium, Miami, FL, while osage copperhead (A.c. phaeogaster) venom was obtained from Dr Hank Guarisco of Lawrence, KS. The F II fractions of these venoms were prepared by carboxymethyl cellulose (Whatman Inc., Clifton, NJ) as previously described (Moran & Geren, 1979b, 1980).

Molecular sieve gels and reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis were purchased from Bio-Rad Laboratories, Richmond, CA. Thrombin-free bovine fibrinogen (9870 clottable) and hide powder azure were obtained from Calbiochem, La Jolla, CA. All other reagents were of analytical quality.

Spectrophotometric determinations were performed with a Gilford Model 252 up-dated Beckman DU. Protein con-

* To whom all correspondence should be addressed.

centrations were estimated by absorbance at 280nm, assuming an extinction coefficient of 1.0 for a 1.0 mg/ml solution.

Sodium dodecyl sulfate polyacrylamide gel electro- phoresis was performed by the method of Weber & Osborn (1968). This procedure was modified as described by Ouyang & Huang (1979) for the separation of fibrino- gen subunits. Coomassie brilliant blue stained electro- phoresis gels were scanned at 600nm with a Gilford spectrophotometer equipped with the linear transport accesory.

Hydrolytic activity against hide powder azure was deter- mined by incubating 5 mg/ml of the substrate in 20mM Tris-buffered normal saline, pH 7.4, with enzyme for 1 hr at

<

L

_k:t ___k3_

i I i i I I I

O I 2 3 4 5 6 7

¢m

Fig. 1. Absorbance scans of sodium dodecyl sulfate electro- phoretic separations of molecular sieve fractions contain- ing fibrinogenase activity. Gels were 5 × 100 mm and 25-30 /ag of protein was applied to each gel. The sharp peak at the right of each gel is due to a pin marking the migration of the bromphenol blue tracking dye. (a) Southern copper- head; (b) broadbanded copperhead: (c) trans-pecos copper-

head; (d) osage copperhead; (e) hemoglobin standard.

349 c B P 70,28 L

350 JEFFREY B. MORAN and COLLIS R. GEREN

37°C. Unsolubilized hide powder was removed with a clini- cal centrifuge and soluble material quantitated by absorb- ance at 595 nm.

The effect of the enzyme on fibrinogen was determined by incubating 5 mg/ml of fibrinogen (98% clottable in l0 mM Tris, pH 7.4, with 10 #g enzyme/ml. Aliquots taken at intervals were used for electrophoresis as described above.

RESULTS

As reported earlier (Moran & Geren, 1980), the fraction F II from the venom of each subspecies of copperhead as obtained by carboxymethyl cellulose ion-exchange chromatography appeared to act on fibrinogen to prevent clot formation. Further separ- ation of the active fractions by molecular sieve chromatography (1 cm × 100cm Bio-Gel P-100 column) produced a protein elution profile identical to that previously reported for northern copperhead venom F II (Moran & Geren, 1981). All of the protei- nase activity of these fractions, as indicated by hide powder azure digestion, coincided with the major protein peak. Figure 1 shows absorbance scans of sodium dodecyl sulfate polyacrylamide electro- phoresis gels of the proteolytic protein peak of each fraction. The scans indicate a prominent band on

o i 2 3 4 5 6 7 8 9 io ClTI

Fig. 2. Effect of the fibrinogenases on fibrinogen subunits as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Bovine fibrinogen (98% clottable, 5 mg/ml in lOmM Tris, pH7.4) was incubated at 25°C with 10/~g/ml of each enzyme. Protein samples of 125/tg were removed from the incubation mixtures at 5 min for electro- phoresis. The sharp peak at the right of each gel is due to a pin marking the migration of the bromphenol blue track- ing due. (a) Southern copperhead; (b) broadbanded copper- head; (c) trans-pecos copperhead; (d) osage copperhead; (e)

non-treated fibrinogen.

each gel corresponding to a mol. wt of 24,000 as com- pared to the migration of a cross-linked hemoglobin standard. This compared favorable to a mol. wt o! 23,000 for each enzyme estimated by elution from the P-100 column which had been calibrated with blue dextran, hemoglobin, myoglobin and potassium ferri- cyanide standards. In the sodium dodecyl sulfate sys- tem, proteins incubated with l j%~ 2-mercaptoethanol co-migrated with the non-reduced protein, indicating that all of these enzymes consist of a single peptide chain.

Figure 2 shows that incubation of fibrinogen (980~i clottable) with the enzyme at 25°C resulted in a rapid disappearance of the ~t-chains of fibrinogen concur- rent with the appearance of a 23,000 tool. wt fragment and a 42,00(04,000 mol. wt band (actually a dimer, similar to that reported for the action of northern copperhead venom on fibrinogen by Moran & Geren, 1981). Incubation of the enzyme with fibrinogen for i hr at 25~C resulted in the disappearance of the 23,000 mol. wt fragment, while the fl and 7+chains ot fibrinogen and the dimer appeared to remain intact.

DISCUSSION

The fibrinogenases described in this report appear to be similar to the fibrinogenases previously isolated and characterized from northern copperhead venom (Moran & Geren, 1981), including similarity in affin- ity for ion-exchange resin, molecular weight and action on fibrinogen. In contrast to phospholipases isolated from copperhead venoms (Moran & Geren, 1980), which have variable properties from one sub- species to another, the fibrinogenase activity appears constant. Thus, the venom of all subspecies of copper- head appears to be a source of a proteinase which has a rapid and specific action on fibrinogen. Such enzymes may prove to be valuable therapeutically and as tools for studying the nature of fibrinogen and other elements of the blood clotting scheme.

Acknowledgements This work was sponsored by NIH Grant GM 24173. C. R. G. currently is the recipient of Research Career Development Award ES 00052.

REFERENCES

MORAN J. B. & GEREN C. R. (1979a) A comparison of biological and chemical properties of three North American (Crotalidae) snake venoms. Toxicon 17, 234-244.

MORAN J. B. ~¢, GEREN C. R. (1979b) Subspecific variations in Agkistrodon contortrix venoms. Comp. Biochem. Phy- siol. 64B, 201-205.

MORAN J. B. & GEREN C. R. (1980) Subspecific variations in Agkistrodon contortrix venoms II. Comp. Biochem. Physiol. 65B, 739-742.

MORAN J. B. & GEREN C. R. (1981) Characterization of a fibrinogenase from northern copperhead venom. Bio- chim. biophys. Acta. in press.

MORAN J. B., PANG S. Y. Y., MARTIN O. W. (~: GEREN C. R. (1979) Separation of northern copperhead venom by ion- exchange chromatography: Preliminary characterization of toxic components. Toxicon 17, 499 510.

MORAN J. B., HILL O. & GEREN C. R. (1981) Comparison of three phospholipases isolated from copperhead venoms. Comp. Biochem. Physiol. in press.

Comparison of copperhead venoms 351

OUYANG C. • TENG C. M. (1976) Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom. Bio- chim. biophys. Acta 420, 298-308.

OUYANG C. & HUANG T. F. (1979) Purification and charac- terization of the fibrinolytic principle of Agkistrodon actus venom. Biochim. biophys. Acta ,139, 146--153.

OUYANG C. & HUANG T. F. (1979) ct- and fl- fibrinogenases from Trimeresurus gramineus snake venom. Biochim. bio- phys. Acta 571,270-283.

OUYANG C., TENG C. M., CHEN Y. C. 8/. LIN S. C. (1978)

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosquamatus venom. Bio- chim. biophys. Acta 541, 394-407.

OUVANG C., TE~qG C. M. & C,EN Y. C. (1979) Properties ol fibrinogen degradation products produced by ~- and fl- fibrinogenases of Trimeresurus mucrosquamatus snake venom. Toxicon 17, 121-126.

WEBER K. & OSBORN M. (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412.