THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 19. Issue of July 5, pp. 8830-8835, 1986 Printed in U S A .

Thrombin-like Enzyme from the Venom of Bitis gubonicu PURIFICATION, PROPERTIES, AND COAGULANT ACTIONS*

(Received for publication, December 9, 1985)

Hubert PirkleS, Ida Theodor, Don Miyada, and Greg Simmons From the Department of Pathology, School of Medicine, University of California, Irvine, California 9271 7

Gabonase, an enzyme which acts on fibrinogen and factor XI11 in uniquely thrombin-like ways, was puri- fied to electrophoretic homogeneity from the venom of Bitis gabonica. On sodium dodecyl sulfate-polyacryl- amide electrophoresis, the reduced protein behaved as a single chain with Mr = 30,600. The enzyme contains 20.6% carbohydrate, no free sulfhydryl groups and hence, from amino acid analysis, five disulfide bonds. Its extinction coefficient (E!:,,,) at 280 nm is 9.6. Its PI is 5.3. Gabonase has an active serine residue, is inac- tivated by phenylmethanesulfonyl fluoride, and has an active histidine which reacts with the chloromethyl ketone of tosyl-L-lysine. Its NHderminal amino acid sequence (Val-Val-Gly-Gly-Ala-Glu-Cys-Lys-Ile-Asp- Gly-His-Arg-Cys-Leu-Ala-Leu-Leu-Tyr-) is homolo- gous to the B chain of thrombin. The activity of the enzyme is stabilized by calcium ion. It exhibits strong Nu-p-tosyl-L-arginine methyl esterase activity, hydro- lyzes tripeptide nitroanilide derivatives weakly or not at all, and cleaves no peptide bonds in insulin, gluca- gon, or the S peptide of ribonuclease. Gabonase clots fibrinogen with a specific activity of 45 NIH thrombin- equivalent unitslmg, releasing both fibrinopeptides A and B and showing substrate inhibition at fibrinogen concentrations of 3 mg/ml or greater. The enzyme also activates factor XIII. It is not inactivated by either heparin or hirudin.

Thrombin is a serine proteinase which catalyzes the last in the series of enzymatic reactions that culminates in the clot- ting of fibrinogen. Thrombin triggers clot formation by cleav- ing FPA’ from the NH, terminus of the Act chain of fibrino- gen. In addition, thrombin releases FPB from the BP chain and activates the fibrin cross-linking enzyme factor XIII. These actions are thought to influence the structure of fibrin and to have an important effect on the vulnerability of fibrin to fibrinolytic degradation (1-3).

* This work was supported in part by Grants HL-13598, HL-22875, and HL-31267 from the National Institutes of Health and the Amer- ican Heart Association, California Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom correspondence should be addressed: Dept. of Pathol- ogy, Medical Sciences I, College of Medicine, Univ. of California, Irvine, CA 92717.

The abbreviations used are: FPA, fibrinopeptide A; FPB, fibrin- opeptide B; Tos, p-toluenesulfonyl; OMe, methoxy; HHTyr, hexahy- drotryosyb pNA, p-nitroanilide; PhGly, phenylglycyb CHGIy, cyclo- hexylglycyl; But, a-aminobutyryb Bz, benzoyl; CHAla, cyclohexyla- lanyl; norleu, norleucyl; Cbo, benzyloxycarbonyl; ,BoGlu, y-(a-t- but0xy)glutamyl; MeO-CO, methoxycarbonyl; SDS, sodium dodecyl sulfate; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatography; PEG, polyethylene glycol.

Whereas the venoms of several snakes contain enzymes that clot fibrinogen by releasing FPA only (4-6) and other thrombin-like venom enzymes preferentially release FPB (7, 8), only an enzyme from the venom of Bitis gabonica, here termed gabonase? has been reported to catalyze, like throm- bin, the activation of factor XI11 as well as the release of both fibrinopeptides (9). Since there are differences among these venom enzymes in their specific thrombin-like actions, their comparative study affords an attractive approach to defining the structural grounds for the coagulant properties that they variously share with thrombin.

In the present work, we describe the isolation from the venom of B. gabonica and the characterization of an enzyme which is uniquely thrombin-like among venom enzymes so far described.

EXPERIMENTAL PROCEDURES AND RESULTS3

RESULTS AND DISCUSSION

Purification of Gabonase from the Venom of B. gabonka- Gabonase was isolated from the crude venom by gel chroma- tography on Sephadex G-200 (Fig. 6A), followed by ion ex- change chromatography on DEAE-cellulose, the latter yield- ing a well-separated peak with constant specific activity (Fig. 6B). The overall purification was about 75-fold, but the actual yield was uncertain due to the apparent removal of interfering material which, by happenstance, gave a calculated yield of 100% (Table 11). The final product was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 8) and by isoelectric focusing. The specific activity of this preparation, using a fibrinogen-clotting assay, was 3.4- 64-fold greater than that of the coagulant fractions separated by Marsh and Whaler (10) and about 1500-fold greater than that reported by Viljoen et al. (ll), possibly reflecting insta- bility of these latter preparations.

Stabilization of enzymatic activity required the presence of calcium ion not only in the crude venom (Fig. 7) but also during purification as well as during storage of the purified protein. Whether calcium protects the enzyme by preventing autolysis (12) or by some other mechanism has not yet been determined.

Physical Properties-By sodium dodecyl sulfate-gel electro-

Marsh and Whaler (35) have proposed the name “gabonase” for the thrombin-like enzyme from the venom of B. gabonica.

Portions of this paper (including “Experimental Procedures,” “Results,” Fig. 6-9, and Tables I1 and 111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-400, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

8830

Thrombin-like Enzyme from Venom of Bitis gabonica 883 1

phoresis, with and without reduction of disulfide bonds, ga- bonase has a single polypeptide chain and an apparent molec- ular weight of about 30,600 (Fig. 8). Gabonase is also homo- geneous by isoelectric focusing, and its PI is 5.3.

The ultraviolet absorption spectrum of gabonase at pH 7.4 shows an asymmetric peak which appears to be a composite of maxima at 276 and 280 nm, a shoulder a t 290 nm, and a minimum at 259 nm, features consistent with the enzyme's aromatic amino acid composition given in Table 111.

Gabonase has an extinction coefficient (E%, at 280 nm) of 9.6.

Chemical Properties-The amino acid compositions given in Table I11 suggest that gabonase, like the other venom enzymes and unlike thrombin, is a fairly acidic protein, and this is borne out by their respective isoelectric points (13-16). In striking contrast to the other enzymes in Table 111, gabon- ase contains only four arginyl bonds and a relatively large number of lysyl bonds, a circumstance that may prove useful in the generation of reversibly blocked tryptic peptides for amino acid sequence analysis. Gabonase is rich in proline, resembling crotalase in that respect. Since gabonase contains no free sulfhydryl groups, all of the cysteine residues found by amino acid analysis must participate in the formation of disulfide bonds.

In common with other snake venom proteins in general (17) and thrombin-like venom enzymes in particular (18,19), gabonase is a glycoprotein with a large carbohydrate compo- nent, 20.6% in total, comprised of sialic acid, mannose, galac- tose, and glucosamine: a composition consistent with a typical complex type of asparagine-linked oligosaccharide (20). Con- trary to the results of Viljoen et al. (ll), fucose was not found.

The NH2-terminal sequence of gabonase is clearly homol- ogous to that of crotalase but less similar to the B chain of thrombin (Fig. 1). However, extended amino acid sequence analysis has shown crotalase to be homologous to the B chain of thrombin (21), so that, on the basis of shared homology, gabonase can be considered homologous to the thrombin B chain (22).

Gabonase resembles thrombin, as well as other blood-clot- ting enzymes, in its narrow specificity of peptide bond cleav- age; it splits no bonds in insulin, glucagon, or the S peptide of ribonuclease. Against tripeptide nitroanilide derivatives as substrates (Table I), gabonase is more fastidious than either thrombin or crotalase (23). Indeed, H-D-CHGly-But-Arg- pNA, an extraordinarily good substrate for thrombin, is among those not hydrolyzed at all by gabonase. On the other hand, Tos-Arg-OMe is a good substrate (Table I).

Active site studies using phenylmethanesulfonyl fluoride and the chloromethyl ketones of tosyllysine and tosylpheny- lalanine (Fig. 9) leave no doubt that gabonase is a serine proteinase. Gabonase is not inactivated by Tos-Phe-CHaC1; on the contrary, it shows a small, transient increase in activity (Fig. 9B), an observation that was confirmed in two separate experiments. We have no obvious explanation for this obser- vation.

Coagulant Actions-The gross physical characteristics of

Gabonase [ T J A D l C l D G v C \ L p -

Crotalase V I G G D E C N I N E H R F L V A L Y -

Thrombin I V E G S N A E I G M S P W Q V M L F -

FIG. 1. NHa-terminal amino acid sequence of gabonase com- pared to crotalase and to thrombin. The thrombin sequence is for the human B chain (34). The crotalase sequence is from Pirkle et al. (21).

TABLE I Kinetic properties of gabonuse with amino acid and peptide amide

and ester substmtes The following amide substrates a t 5 mM were not hydrolyzed H-

D-CHGly-But-Arg-pNA, Bz-Pro-Phe-Arg-pNA, H-D-But-CHAla- Lys-pNA, H-D-norLeu-HHTyr-Lys-pNA, Cbo-BoGlu-Gly-Arg-pNA, and MeO-CO-D-CHGlv-Arrr-DNA.

Substrate" IS1 K, kAe k d K , mM mM s" rnM"s"

Tos-Arg-OMe 0.03-0.5 0.12 12.2 101.7 H-D-Pro-HHTyr-Arg-pNA 0.2-1.6 0.82 0.248 0.30 H-D-PhGly-Phe-Arg-pNA 0.9-3.6 3.40 0.952 (0.28 Tos-Gly-Pro-Arg-pNA 0.1-1.6 0.13 0.014 0.11 Tos-Gly-Pro-Lys-pNA 0.9-3.6 2.72 0.204 0.08 H-D-HHTvr-Ala-Are-DNA 0.9-3.6 0.88 0.065 0.07 ' Tos-kg-OMe reaction mixtures were at pH 8.1 and 30 "C; amide

reaction mixtures were at pH 8.3 and 27 "C.

Time ( m i d FIG. 2. Time course of increasing turbidity of fibrin clots

following addition of gabonase or thrombin. Recorder tracings are shown after addition of enzyme at time 0 to 1.44 mg/ml final concentration of fibrinogen in 0.15 M NaCl, 0.02 M Tris-HC1, pH 7.4.

clots produced by gabonase and by thrombin are entirely similar. However, when clot formation is monitored turbidi- metrically (Fig. 2),4 the time course of increase in turbidity induced by gabonase differs from that induced by thrombin. When the maximal rate of assembly of the two clots is the same, as shown by the steepest part of the sigmoidal curve, the gabonase clot exhibits a much longer lag phase between the addition of enzyme and the first appearance of any tur- bidity. Conversely, when the lag time of the gabonase clot approaches that of the thrombin clot, ita maximal rate of assembly is much greater than that of the thrombin clot. This pattern, i.e. a relatively long lag period followed by rapid assembly of the clot, is typical for the formation of fibrin from monomers which have shed little or no FPB and may reflect the kinetics of total fibrinopeptide charge release (24).

The release of fibrinopeptides by gabonase was studied by gel electrophoresis of the reduced fibrin chains and by HPLC of clot supernatants. Fig. 3 shows the results of an electro- phoretic experiment in which gabonase appeared to convert all of the Aa chain to CY chain and over half of the BB chain to @ chain in 4 h. After 24 h, nearly all of the BP chain had been transformed into a component whose mobility matched the @ chain produced by thrombin. That these transforma-

' Clot turbidity reflects the amount of protein/unit volume incor- porated into the clot and, for a given amount of protein, the thickness of the fibrin fibers (36).

Thrombin-like Enzyme from Venom of Bitis gabonica 8832

Aa

BP, P r Y

a b c d e f Q polymer

ydimer

:P P

FIG. 3. Polyacrylamide gel electrophoresis of reduced fi- brinogen and fibrin chains. Lane a, fibrinogen; lane b, thrombin/ fibrin, 4 h; lane c, gabonase/fibrin, 4 h; lane d, gabonase/fibrin, 24 h; lane e, factor XIII/thrombin/fibrin; lane f, factor XIII/gabonase/ fibrin. Reaction mixtures contained 0.2 mg/ml fibrinogen and 0.27 NIH unit of enzyme. Remaining conditions for clotting and for electrophoresis are given under “Experimental Procedures” and “Re- sults.”

tions were, in fact, the result of fibrinopeptide release was demonstrated by HPLC of clot supernatants (Fig. 4). Thus, the supernatants of gabonase and thrombin clots after 24 h were identical, each showing the release of FPA and its variants and an equivalent amount of FPB. The identity of the small peaks eluted a t 25-26 min is not known, but since they appeared in parallel with FPB and were completely absent when only FPA was released by batroxobin (24), they are most likely related to the desarginine FPB reported by others (25-27). The chromatographic results thus confirm the release of both fibrinopeptides by gabonase and demonstrate that this enzyme cleaves no other peptide from fibrinogen. An HPLC investigation of the time course of fibrinopeptide release (Fig. 5) showed that whereas, as in the case of throm- bin, gabonase releases FPB more slowly than FPA, its release of FPB is even slower than that catalyzed by either human or bovine thrombin.

The optimum concentration of fibrinogen as a substrate for gabonase is narrow. Gabonase clotting times are prolonged not only by fibrinogen concentrations below 1 mg/ml, as was the case with thrombin, but also by fibrinogen concentrations of 3 mg/ml or greater. Thus, for example, when a reaction mixture whose fibrinogen concentration was 6 mg/ml clotted in 29 s using thrombin, an equivalent amount of gabonase produced a clotting time of 40 s. In contrast, thrombin was not inhibited by fibrinogen concentrations up to 6 mg/ml.

The clotting of fibrinogen by gabonase is not inhibited either by hirudin or by heparin, with and without an added source of antithrombin 111. In this respect, gabonase resembles the other thrombin-like venom enzymes, none of which is inactivated by these agents (28).

Gabonase readily activates factor XI11 added to fibrinogen- clotting mixtures, resulting in the formation of electrophoret- ically demonstrable CY chain polymers and the consumption of y chains in the generation of y-y dimers (Fig. 3). Although the venoms of Bothrops moojeni and Agkistrodon rhodostoma also possess factor XIII-activating activity, in neither case is it the property of the thrombin-like enzyme (14, 18). Our observations, along with the evidence that the enzyme from the venom of the Southern copperhead snake, which prefer- entially releases FPB, may also activate factor XI11 (7), might

-Gly-Phe 0

-Gly-Phe B

A

Thrombin

AY

AP

I I I I I I

5 IO 15 20 25 30 Time (mm)

FIG. 4. HPLC patterns of fibrinopeptides released by ga- bonase and by thrombin. Glu-Gly-Phe was added as an internal standard. AP, phosphorylated FPA; A, FPA; AY, desalanine FPA; B, FPB. Conditions for fibrinopeptide release and chromatographic sep- aration are given under “Experimental Procedures” and “Results.”

Time (hr) FIG. 5. Time course of release of FPA and FPB by gabonase

and by human and bovine thrombin. Fibrinopeptides were quan- titated by HPLC. Conditions for fibrinopeptide release and chroma- tography are given under “Experimental Procedures” and “Results.”

gabonase.

raise the question of whether these two activities are somehow linked. Against this possibility, however, is the report that an enzyme from the venom of the Peruvian bushmaster snake releases FPB, as well as FPA, but does not activate factor XI11 (29).

Relationship to Other Studies-Gaffney et al. (9) noted that the venom of B. gabonica contains FPA- and FPB-releasing activities as well as factor XIII-activating activities, but they

_” , FPA; -, FPB; 0, human thrombin; 0, bovine thrombin; X,

Thrombin-like Enzyme from Venom of Bitis gabonica 8833

did not elaborate on these findings. Whether or not these activities were properties of one or more enzymes could not be determined from the partially purified enzyme preparation used by them. The suggestion by these workers that the enzyme had a disulfide-linked subunit structure is not borne out by our results.

Viljoen et ul. (11) isolated from the venom of B. gabonicu an enzyme which resembles gabonase physicochemically and which clots fibrinogen, although at a level of specific activity 1500-fold lower than gabonase. These workers did not exam- ine their enzyme’s ability to release specific fibrinopeptides or to activate factor XIII.

Other studies on the release of fibrinopeptides by venom enzymes have shown that, in addition to those that release FPA only (4-6) and those that release FPB preferentially (7, 8), there are enzymes in the venoms of Trimeresurus okinau- ensis (30) and, as noted, the Peruvian bushmaster snake (29) that release FPA and, in addition, FPB in very small amounts or considerabl~ more slowly. Also, an enzyme from the venom of Agkistrodon halys blomhqffii has been said to release both fibrinopeptides (31), but the details of this work have not been published.

Gabonase does not exhibit any electrophoretically detecta- ble secondary proteolysis of fibrinogen as has been described for crotalase (6), batroxobin, and ancrod (32).

Significunce-the venom analogs of blood-clotting factors offer rich opportunities for structure-function studies. Among thrombin-like enzymes, gabonase is of particular interest because of its exceptional similarity to thrombin with respect to clotting functions examined so far.

Our finding that gabonase, although capable of all of the thrombin-like actions that determine the nature of the fibrin formed, is a single-chain enzyme homologous to only the B chain of thrombin suggests that thrombin’s A chain may not be needed for these actions. Hageman et ai. (33) showed that the B chain of bovine thrombin which had been covalently attached to Sepharose beads is capable of clotting fibrinogen, but they did not determine whether it still could release both fibrinopeptides or activate factor XIII. These questions are now being investigated in our laboratory.

Acknowledgments-We thank Lisa Bundalian, Bina Nandi, and Petar Vukasin for excellent technical assistance; Dr. S. M. Arfin, Dr. E. R. Arquilla, Dr. R. A. Bradshaw, and Dr. F. S. Markland for critical review of the manuscript; and Dr. M. Fukuda and Dr. B. Bothner at the La Jolla Cancer Research F o u n ~ t i o n for carbohydrate analyses.

1.

2. 3.

4. 5.

6. 7.

8.

9.

10.

11.

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Continued on next m e .

8834 Thrombin-like Enzyme from Venom of Bitis gabonica

Froctton Number

I I I I I I 1 2 3 4 5

Time (days)

kDa 13.7 35 25

4.' ' o:, k12 d3 d4 Mobility

Thrombin-like Enzyme f r

24 31

10 14

30 22

7 9

13 16 9

19 23

10

14

20

12

22 12 14

24 20 16

12

17

I 1 I 1

10

13

14 13 8

1 1 17 8 1 1 4 2 5 4

14

21 14 12

7 7 6 8

5 13 6 8

8 9 7 6

17 I 1 10 I 1

4 I 2 18 8

4 6 6

193

16 I 8 1 7

19

- " - 227 267

- 203

-

27

10

15

21

13

23

13

7

18

7

15

21

I 1

9

5

19

18

7

259 -

am Venom of Bitis gabonica 8835