214
~LOOD CLOTTING ENZYMES
[18] T h e C o a g u l a n t E n z y m e f r o m B o t h r o p s Venom (Batroxobin) 1
[18]
atrox
By K. STOCKERand G. H. BA~LOW Bothrops atrox (Linnaeus) is a pit viper which is widespread, and comprises several varieties, in South and Central America. The Linnean species B. atrox has been proposed by Hoge 2 to be subdivided, according to geographical origin, morphological criteria, and immunological properties of their venom, into the five independent species B. asper, B. atrox, B. marajoensis, B. moojeni, and B. pradoi. Further subclassifications of Bothrops atrox have been proposed by several authors; however, a definitive zoological classification is still lacking. A common property of all Bothrops atrox venoms examined in our laboratory is their ability to convert fibrinogen into fibrin, owing to the presence of a thrombinlike enzyme (batroxobin). Assay Methods
Assay on Human Plasma Reagents Citrated human plasma prepared by adding 9 parts freshly drawn venous blood to 1 part sodium citrate solution, 3.8%, and subsequent centrifugation during 30 min at 3000 rpm. A pool obtained from 10 healthy donors is subdivided into 1-ml portions and stored in the frozen state, below --20 ° . Fresh or frozen plasma may be substituted by commercially available citrated normal human plasma (Citrol, Dade, Miami, Florida). Diluent for batroxobin contains sodium chloride, 0.9% chlorobutol, 0.3%, and partially hydrolyzed gelatin 0.02% in distilled water, the pH is adjusted to 6.0. Partial hydrolysis of gelatin is carried out by heating an aqueous solution of gelatin, 2%, pH 3.0, for 30 min at 120° in an autoclave. Method. Citrated human plasma, 0.3 ml, is preincubated for 2 rain at 37 °, in a polystyrene tube 9 X 80 mm, 0.1 ml of batroxobin dilution is added and the time from the enzyme addition to clot formation is reBatroxobin is the generic name for the coagulant fraction from B. atrox venom by the World Health Organization. 2 A. :R. Hoge, Mem. Inst. Butantan Sao Paulo 32, 109 (1965).
COAGULANT ENZYME FROM B. atrox VENOM
[18]
215
corded. Two batroxobin units represent that amount of enzyme, contained in 0.1 ml, which coagulates 0.3 ml of citrated human plasma in 19 ----_0.2 sec. A standard curve is obtained by plotting the log of the clotting time versus the log of the enzyme concentration. The potency of an unknown batroxobin sample can be determined from the graph.
Assay on Bovine Fibrinogen Reagents Bovine fibrinogen, clottability 97-100%, was obtained from Imco, Stockholm. The contents of 1 ampoule is dissolved in 2 ml of distilled water and furnishes a fibrinogen solution, 0.4%, containing 0.15 M Tris-buffer, pH 7.4. Tris-buffer, 0.15 M pH 7.4: Solution A is made by dissolving 1.211 g of Tris and 0.681 g of imidazole in 200 ml of 0.1 N HC1. Solution B is composed of 1.211 g of Tris, 0.681 g of imidazole, and 0.585 g of NaC1 in 300 ml of distilled water. The final solution is prepared by mixing solutions A and B in that proportion to make a final pH of 7.4.
Method. Fibrinogen solution, 0.2 ml, preincubated for 2 min at 37 °, is mixed with 0.2 ml of batroxobin dilution (in Tris buffer, pH 7.4), and the time from the enzyme addition to the clot formation is recorded. A log-log plot is constructed, and unknown activities are calculated from the curve. Quantitative Estimation o] Batroxobin in Serum (Micromethod) Principle. Whereas thrombin is adsorbed and thereby inactivated by fibrin, the activity of batroxobin is not affected by fibrin. If batroxobincontaining serum, e.g., serum of a patient undergoing defibrinogenating therapy with Defibrase, is overlayered by a fibrinogen solution, a clot starts to grow at the interface and reaches a length that is proportional to the batroxobin concentration in the serum. Reagents Fibrinogen solution, 0.2%, prepared by dissolving the content of one ampoule of bovine fibrinogen, Imco, Stockholm, in 4 ml of propylene glycol 2.5%, in distilled water Heparinized sermn: normal human serum or patient serum, containing 50 units of heparin per milliliter. Batroxobin dilutions are prepared by diluting Defibrase, 20 batroxobin units (BU) ml (Pentapharm Ltd., CH-4002, Basle, Switzerland) with heparinized normal serum.
216
BLOOD CLOTTING ENZYMES
[18]
Method. Batroxobin dilution in heparinized serum, 0.1 ml, is carefully filled into glass tubes, 4 )~ 60 mm, by means of a syringe, and subsequently overlayered with 0.3 ml of fibrinogen solution, 0.2%. The tubes are kept in a humid atmosphere, at 30 °, for 15 hr (overnight) in order to allow clot propagation (Fig. 1). The length of each clot is measured by means of a micrometer device and recorded on a semilogarithmic scale as a function of the batroxobin concentration in the serum. From this standard curve the batroxobin concentration in a patient serum may be estimated. Assay on BZ-L-Phe-L-Val-L-Arg-pNA Principle. Hydrolysis of benzoylphenylalanylvalylarginine p-nitroanilide by batroxobin liberates a quantity of p-nitroaniline proportional to the enzyme activity. The liberation of p-nitroaniline is measured by following the change in absorbance at 405 nm.
Fro. 1. S t a t e of clots i n d u c e d b y o v e r l a y e r i n g b a t r o x o b i n d i l u t i o n s in n o r m a l h u m a n serum, w i t h b o v i n e fibrinogen, 0.4%. B a t r o x o b i n c o n c e n t r a t i o n s per m i l l i l i t e r of s e r u m : 1 = 0.0005 B U , 2 = 0.001 B U , 3 = 0.002 B U , 4 = 0.004 B U , 5 = 0.008 B U , 6 = 0.016 B U , 7 = 0.032 B U , 8 = 0.064 B U . B U = b a t r o x o b i n units.
COAGULANT ENZYME FROM B. atrox VENOM
[18]
217
Reagents Bz-Phe-Val-Arg-pNA-HC1, substrate S-2160, is commercially available through AB Bofors, Nobel Division, Peptide Research, S-431, ss Molndal, Sweden. A 1 m M solution is prepared by dissolving 0.68 g of substrate in 1 ml of distilled water. Tris-imidazole buffer, 0.15 M, pH 8.2, prepared as follows: solution A contain 1.211 g Tris and 0.681 g of imidazole in 200 ml of HC1 0.1 M; solution B is composed of 1.211 g of Tris, 0.681 g of imidazole, and 0.585 g of NaC1 in 300 ml of distilled water. Solutions A and B are mixed in such a proportion so as to make a final pH of 8.2.
Method. Buffer, 2.00 ml, and batroxobin solution, 0.25 ml, are preincubated for 2 min at 37 °, in a 1-cm cuvette; 0.25 ml of substrate solution is added, and adsorbance is recorded during 2 min. Calculation. Abs./min )< 1000 = mU/ml (1 mU of batroxobin converts 1 t~mole of substrate per minute). Qualitative Detection of Batroxobin in lmmunopherograms ~ Principle. hnmunopherograms of either crude B. atroz venoms or of batroxobin preparations are overlaid by a fibrinogen-agarose gel. Fibrin is formed at the site of the thrombinlike enzyme in the pherogram and allows the identity of the corresponding precipitin line and the estimation of eleetrophoretic mobility of batroxobin. Reagents Barbital buffer, pH 8.6, containing 3.7 g of barbital and 20.6 g of barbital sodium in 2 liters of distilled water Staining solution containing 500 mg of Amidoschwarz (Merck, Darmstadt, GFR) in a mixture composed of 50 ml of acetic acid and 500 ml of ethanol Destaining solution, containing 10% acetic acid in ethanol Agarose plates, 26 X 76 ram, prepared from a solution of 200 mg of agarose in 20 ml of barbital buffer, pH 8.6 Fibrinogen-agarose plates, 26 X 76 mm, prepared as follows: 100 mg of agarose are dissolved in 10 ml of distilled water at 95 ° and cooled to 40 °. 10 ml of bovine fibrinogen, 0.4% (Imco, Stockholm, Sweden) are heated to 40 ° . Both solutions are mixed and immediately transferred, in 2-ml portions, onto the surface of the glass plates and allowed to gel. '~K. Stoeker, W. Christ, and P. Leloup, Toxicon 12, 415 (1974).
218
BLOOD CLOTTING ENZYMES
[18]
Polyvalent anti-Bothrops serum (horse), commercially available through Inst. Butantan, Sao Paulo, Brazil or through Lab. Behrens, Caracas, Venezuela. Method. Immunoeleetrophoresis is carried out by conventional techniques in 1% agarose, pH 8.6, 40 V/cm, with 5 ~l of samples containing 1-10 mg of protein per milliliter. Immunodiffusion is performed in humid atmosphere for 18 hr after filling the central trough with 20 ~l of antiserum. Intergel-reaction with fibrinogen-agarose is performed by covering the pherogram after immunodiffusion with a fibrinogen-agarose plate and allowing them to react until a distinct turbidity caused by fibrin appears (5-15 min at room temperature). The superposed fibrinogen-agarose gel is cautiously removed and discarded. Excess of fibrinogen, serum and venom proteins is eliminated b:~ washing the pherograms for 6 hr in 0.15 M NaC1. The moist gels are covered with humidified filter paper and dried in an air stream. The filter paper is removed from the gel surface after a brief dip in distilled water and the pherograms are kept for 5 min in the staining solution and subsequently destained by three treatments with destaining solution, washed in 10% acetic acid, and finally rinsed in distilled water. The gels are allowed to dry at room temperature. By this procedure, precipitin lines are revealed as in normal immunoelectrophoresis and thrombinlike activity appears in addition as blue "shadow" of stained fibrin (Fig. 2). Purification
The purification of batroxobin is achieved by chromatography of the crude B. atrox venom on DEAE-Sephadex and subsequent rechromatography of the coagulant fraction on Sephadex G-I(D. Chromatography on DEAE-Sephadex. DEAE-Sephadex A-50 (AB Pharmacia, Uppsala. Sweden) is equilibrated with Tris-phosphate buffer, pH 6.0, 0.07 M, and packed into a column, 60 X 600 mm, equipped for ascending chromatography. One gram of B. atrox venom is dissolved in 20 ml of Tris-phosphate buffer, pH 6.0, 0.07 M, and applied to the column. The elution is carried out using the same Tris-phosphate buffer; optical density is recorded at 280 nm and fractions of 15 ml are collected. The batroxobin-containing fractions are identified by determination of the clotting time of 0.2 ml of bovine fibrinogen 0.4% after addition of 0.2 ml of eluate. The batroxobin-containing fractions are pooled together, and the buffer is removed by ultrafiltration through a UM-2 Diaflo membrane (Amicon Corp., Lexington, Massachusetts), and several washings with
[18]
COAGULANT ENZYME FROM B. atrox VENOM
219
Fro. 2. Immunopherograms with localized batroxobin activity of the crude venoms of five zoologicalvarieties of Bothrops atrox.
ammonium formate solution, 1%. The Tris-phosphate free eluate is finally concentrated on the ultrafilter to one-tenth of its initial volume and freeze dried. Chromatography on Sephadex G-IO0. One hundred milligrams of freeze-dried, desalted batroxobin containing eluate from the chromatography on DEAE-Sephadex are dissolved in 0.5 ml of distilled water and applied to a column, 30 X 100 mm, of Sephadex G-100, previously equilibrated with the solvent composed of tertiary butanol 10% and ammonium formate 1% in distilled water, pH 4.0 (adjusted with NH~OH). Descending chromatography is carried out using the above solvent system; absorbancy of the eluate is recorded at 280 nm, and fractions of 5 ml are collected. The batroxobin-containing fractions are identified by measuring the clotting time of 0.2 ml of bovine fibrinogen, 0.4%, after addition of 0.2 ml of eluate, previously diluted in the ratio 1:5 with ammonium bicarbonate solution, 1%. The pooled active eluate is freeze-dried. Almost salt-free, electrophoretically homogeneous batroxobin is obtained. Ammonium formate may be completely removed by dialysis of batroxobin against 0.1 M acetic acid and subsequent lyophilization.
220
[18]
BLOOD CLOTTING ENZYMES
TABLE I SPECIFIC ACTIVITY OF BATROXOBIN ISOLATED FROM VENOM OF THREE DIFFERENT VARIETIES OF Bothrops atrox
Variety
Batrexobin units/mg
B. asper B. marajoensis B. moojeni
2000 1900 500
Properties Purity. Batroxobin isolated following the above method from the venom of one single variety of Bothrops atrox shows one precipitin line on immunoelectrophoresis against polyvalent anti-Bothrops serum, it also gives one single band in electrophoresis on polyacrylamide gel, 7.5%, pH 2.5. Electrophoresis of the previously reduced and alkylated enzyme in sodium dodecyl sulfate (SDS) containing polyacrylamide, 7%, shows also one single band after staining with either Coomassie blue (peptide moiety) or Schiff's reagent (carbohydrate moiety). The specific activity of batroxobin deriving from the venom of various subspecies of Bothrops atrox shows substantial differences (Table I). Physical Properties. As shown in Fig. 2, the thrombinlike enzyme of different varieties of Bothrops atrox migrate with different electrophoretic mobility in agarose, 1%, pH 8.6. Similar differences are also observed in electrophoresis in polyacrylamide gel, even in the presence of SDS, which indicate differences in molecular weight. Differences in the molecular weights of batroxobin originating from B. marajoensis and B. moojeni venom have been confirmed by ultracentrifugation (Table II). TABLE II MOLECULAR WEIGHT OF BATROXOBIN ISOLATED FROM DIFFERENT ZOOLOGICAL VARIETIES OF Bothrops atrox
Molecular weight Batroxobin from
SDS ~ electrophoresis
Ultracentrifugation
B. asper B. marajoensis B. moojeni
32,000 41,500 36,000
43,000 37,000
a SDS, sodium dodecyl sulfate.
[18]
COAGULANT
ENZYME
FROM
B. alrox
VENOM
221
The isoeleetrie point of batroxobin from B. moojeni venom is 6.6, as determined by isoelectric focusing, c~r~l% ~ 1 cm of batroxobin from B. moojeni venom was found to be 10.5. Chemical Properties. Reduced and alkylated batroxobin shows in SDS eleetrophoresis, after staining with either Coomassie blue or Schiff's reagent, one single band that characterizes batroxobin as a single-chain glycopeptide. The neutral carbohydrate content of batroxobin from B. marajoensis venom is 10.2% whereas batroxobin from B. moojeni venom contains only 5.8% of neutral sugar. Extensive treatment of batroxobin with a-neuraminidase reduces the specific activity of the enzyme from B. marajoensis by 52% and of the enzyme from B. moojeni venom by 39%. The electrophoretic mobility in polyacrylamide gel, 7.5%, pH 2.5, of neuraminidase-treated batroxobin is significantly increased, whereas the mobility in immunoelectrophoresis in agarose 1%, pH 8.6 is decreased. The NH~-terminal amino acid residue of batroxobin from either B. marajoensis or B. moojeni venom is valine. Immunological Properties. Batroxobin is antigenic, and specific antibody preparations may be obtained from animals immunized by periodical intramuscular injections. The specific rabbit antisera against batroxobin prepared from (a) B. marajoensis venom or (b) B. moojeni venom are able to neutralize the clotting activity as well as the amidolytic activity on Bz-Phe-Val-Arg-pNA of the crude venoms from B. asper,
B. atrox columbiensis, B. atrox venezolensis, B. marajoensis, B. moojeni, and B. pradoi. The clotting activity of Agkistrodon rhodostoma venom, however, is not neutralized by these antibody preparations, a fact that confirms previous immunodiffusion experiments. 4,5 Stability. Batroxobin isolated from either B. moojeni or B. marajoensis venom remains stable in aqueous solutions in the wide pH range of 2.5-9 for several hours at 20% A solution of batroxobin in glycerol may be heated for 1 hr at 100 ° without significant loss of activity. Dilute solutions (20 BU/ml) in physiological saline, pH 6, containing 0.02% of gelatin and 0.3% of chlorobutol have remained stable for more than a year at +4% Repeated freezing and thawing does not affect the activity of batroxobin solutions. Specificity. Batroxobin converts fibrinogen into fibrin by specific cleavage of those Arg-Gly bonds in the a-chain, which leads to release of 4 G. It. Barlow, L. J. Lewis, R. Finlay, D. Martin, and K. Stocker, Thromb. Res. 2, 17 (1973). 5 K. Stoeker and G. H. Barlow in "Defibrinierung mit thrombinghnliehen Sehlangengiftenzymen" (M. Martin and W. Schoop, eds.), pp. 45-63. Huber, Bern, 1974.
222
BLOOD CLOTTING ENZYMES
[18]
fibrinopeptide A, whereas the fl-chain of fibrinogen remains unaffected2 ,7 Prolonged incubation of fibrin with batroxobin causes, just as does t h a t of thrombin, release of the tripeptide Gly-Pro-Arg. 8 Batroxobin neither exerts a direct fibrinolytic activity nor activates plasminogen. Batroxobin acts with a species-dependent preference on fibrinogen of different m a m m a l s " ; hence, coagulation of r a b b i t fibrinogen takes about 10 times longer t h a n the coagulation of h u m a n fibrinogen after additions of equal amounts of batroxobin. This phenomenon might be explained b y structural differences of rabbit and h u m a n fibrinogen as reflected in the sequence analysis of fibrinopeptide A from different mammals. 1° I n contrast to thrombin, batroxobin does not activate h u m a n factor V I I I 11 and neither induces nor affects the platelet's aggregation and release reactions. 12 Therefore, it forms a nonretracting clot with plateletrich h u m a n plasma. ~3 Batroxobin isolated from the venoms of B. asper and B. moojeni, like thrombin, activates factor X I I I , whereas batroxobin from B. marajoensis venom does not activate this factor. 14 Thus, batroxobin from B. asper and B. mo,ojeni venom, in the presence of Ca 2+, forms with prothrombin-free h u m a n plasma, a clot t h a t remains insoluble in 5 M urea or 1% monochloroacetic acid, whereas batroxobin from B. marajoensis forms a clot t h a t is rapidly dissolved in these agents. Batroxobin is able to hydrolyze arginine esters such as N"-benzoyl-L arginine ethyl ester or toluenesulfonyl-L-arginine methyl ester, 15 and it furthermore digests the synthetic fibrinopeptide A analogs B z - P h e - V a l A r g - p N A and d a n s y l - G l y - G l y - V a l - A r g - G l y O M E . 16 Batroxobin originating from the different varieties of Bothrops atrox acts with a different velocity on B z - P h e - V a l - A r g - p N A (Table I I I ) . Activators and Inactivators. Batroxobin-induced coagulation of fibrinogen is accelerated in the presence of imidazole or phenol. Since 6B. Blombiick, M. Blomb~ick, and I. M. Nilsson, Thromb. Diath. Haemorrh. 1, 76 (1957). ' K. Stocker and P. W. Straub, Thromb. Diath. Haemorrh. 24, 248 (1970). SB. ttessel and M. Blomb~ick, FEBS Left. 18, 318 (1971). 9 K. Wik, O. Tangen, and F. McKenzie, Br. ]. Haematol. 23, 37 (1972). lo K. Laki, Fibrinogen 1968, 9 (1968). "S. Lopaciuk and Z. S. Latallo, Abstr. Int. Congr. Thromb. Haematol, $th (1973). 12S., Niewiarowski, J. St. Stewart, N. Nath, A. Tai Sha, and G. E. Liebermann, Am. J. Physiol. in press 18G. DeGaetano, R. Franco, M. B. Donati, A. Bonaccorsi, and S. Garattini, Thromb. Res. 4, 189 (1974). ~ J. McDonagh and R. P. McDonagh, Abstr. Int. Congr. Thromb. Haematol., $th (1973). ,5 j. Soria, C. Soria, J. Yver, and M. Samama, Coagulation 2, 173 (1969). aeK. H. Chen, I. Simpson, J. Bruner-Lorand, and L. Lorand Abstr. Meet. Fed. Am. Soc. Exp. Biol. (1972).
[19]
CROTALASE
223
TABLE III ACTION OF BATROXOBINORIGINATING FROMBothrops marajoensis AND B. moojeni ON Bz-PuE-VAL-ARG-pNA Vm~ (inoles/min) Batroxobin from
K (M)
Per mU
Per BU"
B. marajoensis B. moojeni
1.61 X 10-4 2.86 X 10-4
2 3.9
2.38 X 10-3 0.59 X 10-3
a BU = batroxobin unit. hydrolysis of Bz-Phe-Val-Arg-pNA is also potentiated by imidazole but not by phenol, imidazole m a y be considered as an activator, whereas phenol seems to catalyze fibrin polymerization. Batroxobin is inactivated by 2.5 m M diisopropyl fiuorophosphate, pH 8. In contrast to thrombin, it is not inactivated by incubation for 15 hr at 20 ° in iodoacetamide, 0.1%, pH 7.4, and, whereas thrombin looses its clotting activity after 1 hr of incubation in SDS 0.01%, pH 7, at 20 °, this treatment does not affect the activity of batroxobin. Batroxobin is inactivated neither by thrombin inhibitors, such as heparin, heparinoids, or hirudin, nor by proteinase inhibitors, such as aprotinin, soybean inhibitor, nor by plasmin inhibitors such as e-aminocaproic acid or tranexamic acid. Batroxobin is bound to a2-macroglobulin and looses its clotting activity, whereas its amidolytic activity on the small synthetic substrate Bz-Phe-Val-Arg-pNA remains almost unaffected. 17 1, N. Egberg, Thromb. Res. 4, 35 (1974).
[19] Crotalase B y FRANCIS S. MARKLAND,JR. Crotalase is a thrombinlike enzyme isolated from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). 1 In 1886, Mitchell and Reichert 2 demonstrated that animal blood failed to coagulate after treatment with C. adamanteus venom. They carried out extensive studies on this venom and showed that the globulin (protein) F. S. Markland and P. S. Damus, d. Biol. Chem. 264, 6460 (1971). 2S. W. Mitchell and E. T. Reichert, Smithsonian Contrib. Knowl. 26, 1 (1886).
~LOOD CLOTTING ENZYMES
[18] T h e C o a g u l a n t E n z y m e f r o m B o t h r o p s Venom (Batroxobin) 1
[18]
atrox
By K. STOCKERand G. H. BA~LOW Bothrops atrox (Linnaeus) is a pit viper which is widespread, and comprises several varieties, in South and Central America. The Linnean species B. atrox has been proposed by Hoge 2 to be subdivided, according to geographical origin, morphological criteria, and immunological properties of their venom, into the five independent species B. asper, B. atrox, B. marajoensis, B. moojeni, and B. pradoi. Further subclassifications of Bothrops atrox have been proposed by several authors; however, a definitive zoological classification is still lacking. A common property of all Bothrops atrox venoms examined in our laboratory is their ability to convert fibrinogen into fibrin, owing to the presence of a thrombinlike enzyme (batroxobin). Assay Methods
Assay on Human Plasma Reagents Citrated human plasma prepared by adding 9 parts freshly drawn venous blood to 1 part sodium citrate solution, 3.8%, and subsequent centrifugation during 30 min at 3000 rpm. A pool obtained from 10 healthy donors is subdivided into 1-ml portions and stored in the frozen state, below --20 ° . Fresh or frozen plasma may be substituted by commercially available citrated normal human plasma (Citrol, Dade, Miami, Florida). Diluent for batroxobin contains sodium chloride, 0.9% chlorobutol, 0.3%, and partially hydrolyzed gelatin 0.02% in distilled water, the pH is adjusted to 6.0. Partial hydrolysis of gelatin is carried out by heating an aqueous solution of gelatin, 2%, pH 3.0, for 30 min at 120° in an autoclave. Method. Citrated human plasma, 0.3 ml, is preincubated for 2 rain at 37 °, in a polystyrene tube 9 X 80 mm, 0.1 ml of batroxobin dilution is added and the time from the enzyme addition to clot formation is reBatroxobin is the generic name for the coagulant fraction from B. atrox venom by the World Health Organization. 2 A. :R. Hoge, Mem. Inst. Butantan Sao Paulo 32, 109 (1965).
COAGULANT ENZYME FROM B. atrox VENOM
[18]
215
corded. Two batroxobin units represent that amount of enzyme, contained in 0.1 ml, which coagulates 0.3 ml of citrated human plasma in 19 ----_0.2 sec. A standard curve is obtained by plotting the log of the clotting time versus the log of the enzyme concentration. The potency of an unknown batroxobin sample can be determined from the graph.
Assay on Bovine Fibrinogen Reagents Bovine fibrinogen, clottability 97-100%, was obtained from Imco, Stockholm. The contents of 1 ampoule is dissolved in 2 ml of distilled water and furnishes a fibrinogen solution, 0.4%, containing 0.15 M Tris-buffer, pH 7.4. Tris-buffer, 0.15 M pH 7.4: Solution A is made by dissolving 1.211 g of Tris and 0.681 g of imidazole in 200 ml of 0.1 N HC1. Solution B is composed of 1.211 g of Tris, 0.681 g of imidazole, and 0.585 g of NaC1 in 300 ml of distilled water. The final solution is prepared by mixing solutions A and B in that proportion to make a final pH of 7.4.
Method. Fibrinogen solution, 0.2 ml, preincubated for 2 min at 37 °, is mixed with 0.2 ml of batroxobin dilution (in Tris buffer, pH 7.4), and the time from the enzyme addition to the clot formation is recorded. A log-log plot is constructed, and unknown activities are calculated from the curve. Quantitative Estimation o] Batroxobin in Serum (Micromethod) Principle. Whereas thrombin is adsorbed and thereby inactivated by fibrin, the activity of batroxobin is not affected by fibrin. If batroxobincontaining serum, e.g., serum of a patient undergoing defibrinogenating therapy with Defibrase, is overlayered by a fibrinogen solution, a clot starts to grow at the interface and reaches a length that is proportional to the batroxobin concentration in the serum. Reagents Fibrinogen solution, 0.2%, prepared by dissolving the content of one ampoule of bovine fibrinogen, Imco, Stockholm, in 4 ml of propylene glycol 2.5%, in distilled water Heparinized sermn: normal human serum or patient serum, containing 50 units of heparin per milliliter. Batroxobin dilutions are prepared by diluting Defibrase, 20 batroxobin units (BU) ml (Pentapharm Ltd., CH-4002, Basle, Switzerland) with heparinized normal serum.
216
BLOOD CLOTTING ENZYMES
[18]
Method. Batroxobin dilution in heparinized serum, 0.1 ml, is carefully filled into glass tubes, 4 )~ 60 mm, by means of a syringe, and subsequently overlayered with 0.3 ml of fibrinogen solution, 0.2%. The tubes are kept in a humid atmosphere, at 30 °, for 15 hr (overnight) in order to allow clot propagation (Fig. 1). The length of each clot is measured by means of a micrometer device and recorded on a semilogarithmic scale as a function of the batroxobin concentration in the serum. From this standard curve the batroxobin concentration in a patient serum may be estimated. Assay on BZ-L-Phe-L-Val-L-Arg-pNA Principle. Hydrolysis of benzoylphenylalanylvalylarginine p-nitroanilide by batroxobin liberates a quantity of p-nitroaniline proportional to the enzyme activity. The liberation of p-nitroaniline is measured by following the change in absorbance at 405 nm.
Fro. 1. S t a t e of clots i n d u c e d b y o v e r l a y e r i n g b a t r o x o b i n d i l u t i o n s in n o r m a l h u m a n serum, w i t h b o v i n e fibrinogen, 0.4%. B a t r o x o b i n c o n c e n t r a t i o n s per m i l l i l i t e r of s e r u m : 1 = 0.0005 B U , 2 = 0.001 B U , 3 = 0.002 B U , 4 = 0.004 B U , 5 = 0.008 B U , 6 = 0.016 B U , 7 = 0.032 B U , 8 = 0.064 B U . B U = b a t r o x o b i n units.
COAGULANT ENZYME FROM B. atrox VENOM
[18]
217
Reagents Bz-Phe-Val-Arg-pNA-HC1, substrate S-2160, is commercially available through AB Bofors, Nobel Division, Peptide Research, S-431, ss Molndal, Sweden. A 1 m M solution is prepared by dissolving 0.68 g of substrate in 1 ml of distilled water. Tris-imidazole buffer, 0.15 M, pH 8.2, prepared as follows: solution A contain 1.211 g Tris and 0.681 g of imidazole in 200 ml of HC1 0.1 M; solution B is composed of 1.211 g of Tris, 0.681 g of imidazole, and 0.585 g of NaC1 in 300 ml of distilled water. Solutions A and B are mixed in such a proportion so as to make a final pH of 8.2.
Method. Buffer, 2.00 ml, and batroxobin solution, 0.25 ml, are preincubated for 2 min at 37 °, in a 1-cm cuvette; 0.25 ml of substrate solution is added, and adsorbance is recorded during 2 min. Calculation. Abs./min )< 1000 = mU/ml (1 mU of batroxobin converts 1 t~mole of substrate per minute). Qualitative Detection of Batroxobin in lmmunopherograms ~ Principle. hnmunopherograms of either crude B. atroz venoms or of batroxobin preparations are overlaid by a fibrinogen-agarose gel. Fibrin is formed at the site of the thrombinlike enzyme in the pherogram and allows the identity of the corresponding precipitin line and the estimation of eleetrophoretic mobility of batroxobin. Reagents Barbital buffer, pH 8.6, containing 3.7 g of barbital and 20.6 g of barbital sodium in 2 liters of distilled water Staining solution containing 500 mg of Amidoschwarz (Merck, Darmstadt, GFR) in a mixture composed of 50 ml of acetic acid and 500 ml of ethanol Destaining solution, containing 10% acetic acid in ethanol Agarose plates, 26 X 76 ram, prepared from a solution of 200 mg of agarose in 20 ml of barbital buffer, pH 8.6 Fibrinogen-agarose plates, 26 X 76 mm, prepared as follows: 100 mg of agarose are dissolved in 10 ml of distilled water at 95 ° and cooled to 40 °. 10 ml of bovine fibrinogen, 0.4% (Imco, Stockholm, Sweden) are heated to 40 ° . Both solutions are mixed and immediately transferred, in 2-ml portions, onto the surface of the glass plates and allowed to gel. '~K. Stoeker, W. Christ, and P. Leloup, Toxicon 12, 415 (1974).
218
BLOOD CLOTTING ENZYMES
[18]
Polyvalent anti-Bothrops serum (horse), commercially available through Inst. Butantan, Sao Paulo, Brazil or through Lab. Behrens, Caracas, Venezuela. Method. Immunoeleetrophoresis is carried out by conventional techniques in 1% agarose, pH 8.6, 40 V/cm, with 5 ~l of samples containing 1-10 mg of protein per milliliter. Immunodiffusion is performed in humid atmosphere for 18 hr after filling the central trough with 20 ~l of antiserum. Intergel-reaction with fibrinogen-agarose is performed by covering the pherogram after immunodiffusion with a fibrinogen-agarose plate and allowing them to react until a distinct turbidity caused by fibrin appears (5-15 min at room temperature). The superposed fibrinogen-agarose gel is cautiously removed and discarded. Excess of fibrinogen, serum and venom proteins is eliminated b:~ washing the pherograms for 6 hr in 0.15 M NaC1. The moist gels are covered with humidified filter paper and dried in an air stream. The filter paper is removed from the gel surface after a brief dip in distilled water and the pherograms are kept for 5 min in the staining solution and subsequently destained by three treatments with destaining solution, washed in 10% acetic acid, and finally rinsed in distilled water. The gels are allowed to dry at room temperature. By this procedure, precipitin lines are revealed as in normal immunoelectrophoresis and thrombinlike activity appears in addition as blue "shadow" of stained fibrin (Fig. 2). Purification
The purification of batroxobin is achieved by chromatography of the crude B. atrox venom on DEAE-Sephadex and subsequent rechromatography of the coagulant fraction on Sephadex G-I(D. Chromatography on DEAE-Sephadex. DEAE-Sephadex A-50 (AB Pharmacia, Uppsala. Sweden) is equilibrated with Tris-phosphate buffer, pH 6.0, 0.07 M, and packed into a column, 60 X 600 mm, equipped for ascending chromatography. One gram of B. atrox venom is dissolved in 20 ml of Tris-phosphate buffer, pH 6.0, 0.07 M, and applied to the column. The elution is carried out using the same Tris-phosphate buffer; optical density is recorded at 280 nm and fractions of 15 ml are collected. The batroxobin-containing fractions are identified by determination of the clotting time of 0.2 ml of bovine fibrinogen 0.4% after addition of 0.2 ml of eluate. The batroxobin-containing fractions are pooled together, and the buffer is removed by ultrafiltration through a UM-2 Diaflo membrane (Amicon Corp., Lexington, Massachusetts), and several washings with
[18]
COAGULANT ENZYME FROM B. atrox VENOM
219
Fro. 2. Immunopherograms with localized batroxobin activity of the crude venoms of five zoologicalvarieties of Bothrops atrox.
ammonium formate solution, 1%. The Tris-phosphate free eluate is finally concentrated on the ultrafilter to one-tenth of its initial volume and freeze dried. Chromatography on Sephadex G-IO0. One hundred milligrams of freeze-dried, desalted batroxobin containing eluate from the chromatography on DEAE-Sephadex are dissolved in 0.5 ml of distilled water and applied to a column, 30 X 100 mm, of Sephadex G-100, previously equilibrated with the solvent composed of tertiary butanol 10% and ammonium formate 1% in distilled water, pH 4.0 (adjusted with NH~OH). Descending chromatography is carried out using the above solvent system; absorbancy of the eluate is recorded at 280 nm, and fractions of 5 ml are collected. The batroxobin-containing fractions are identified by measuring the clotting time of 0.2 ml of bovine fibrinogen, 0.4%, after addition of 0.2 ml of eluate, previously diluted in the ratio 1:5 with ammonium bicarbonate solution, 1%. The pooled active eluate is freeze-dried. Almost salt-free, electrophoretically homogeneous batroxobin is obtained. Ammonium formate may be completely removed by dialysis of batroxobin against 0.1 M acetic acid and subsequent lyophilization.
220
[18]
BLOOD CLOTTING ENZYMES
TABLE I SPECIFIC ACTIVITY OF BATROXOBIN ISOLATED FROM VENOM OF THREE DIFFERENT VARIETIES OF Bothrops atrox
Variety
Batrexobin units/mg
B. asper B. marajoensis B. moojeni
2000 1900 500
Properties Purity. Batroxobin isolated following the above method from the venom of one single variety of Bothrops atrox shows one precipitin line on immunoelectrophoresis against polyvalent anti-Bothrops serum, it also gives one single band in electrophoresis on polyacrylamide gel, 7.5%, pH 2.5. Electrophoresis of the previously reduced and alkylated enzyme in sodium dodecyl sulfate (SDS) containing polyacrylamide, 7%, shows also one single band after staining with either Coomassie blue (peptide moiety) or Schiff's reagent (carbohydrate moiety). The specific activity of batroxobin deriving from the venom of various subspecies of Bothrops atrox shows substantial differences (Table I). Physical Properties. As shown in Fig. 2, the thrombinlike enzyme of different varieties of Bothrops atrox migrate with different electrophoretic mobility in agarose, 1%, pH 8.6. Similar differences are also observed in electrophoresis in polyacrylamide gel, even in the presence of SDS, which indicate differences in molecular weight. Differences in the molecular weights of batroxobin originating from B. marajoensis and B. moojeni venom have been confirmed by ultracentrifugation (Table II). TABLE II MOLECULAR WEIGHT OF BATROXOBIN ISOLATED FROM DIFFERENT ZOOLOGICAL VARIETIES OF Bothrops atrox
Molecular weight Batroxobin from
SDS ~ electrophoresis
Ultracentrifugation
B. asper B. marajoensis B. moojeni
32,000 41,500 36,000
43,000 37,000
a SDS, sodium dodecyl sulfate.
[18]
COAGULANT
ENZYME
FROM
B. alrox
VENOM
221
The isoeleetrie point of batroxobin from B. moojeni venom is 6.6, as determined by isoelectric focusing, c~r~l% ~ 1 cm of batroxobin from B. moojeni venom was found to be 10.5. Chemical Properties. Reduced and alkylated batroxobin shows in SDS eleetrophoresis, after staining with either Coomassie blue or Schiff's reagent, one single band that characterizes batroxobin as a single-chain glycopeptide. The neutral carbohydrate content of batroxobin from B. marajoensis venom is 10.2% whereas batroxobin from B. moojeni venom contains only 5.8% of neutral sugar. Extensive treatment of batroxobin with a-neuraminidase reduces the specific activity of the enzyme from B. marajoensis by 52% and of the enzyme from B. moojeni venom by 39%. The electrophoretic mobility in polyacrylamide gel, 7.5%, pH 2.5, of neuraminidase-treated batroxobin is significantly increased, whereas the mobility in immunoelectrophoresis in agarose 1%, pH 8.6 is decreased. The NH~-terminal amino acid residue of batroxobin from either B. marajoensis or B. moojeni venom is valine. Immunological Properties. Batroxobin is antigenic, and specific antibody preparations may be obtained from animals immunized by periodical intramuscular injections. The specific rabbit antisera against batroxobin prepared from (a) B. marajoensis venom or (b) B. moojeni venom are able to neutralize the clotting activity as well as the amidolytic activity on Bz-Phe-Val-Arg-pNA of the crude venoms from B. asper,
B. atrox columbiensis, B. atrox venezolensis, B. marajoensis, B. moojeni, and B. pradoi. The clotting activity of Agkistrodon rhodostoma venom, however, is not neutralized by these antibody preparations, a fact that confirms previous immunodiffusion experiments. 4,5 Stability. Batroxobin isolated from either B. moojeni or B. marajoensis venom remains stable in aqueous solutions in the wide pH range of 2.5-9 for several hours at 20% A solution of batroxobin in glycerol may be heated for 1 hr at 100 ° without significant loss of activity. Dilute solutions (20 BU/ml) in physiological saline, pH 6, containing 0.02% of gelatin and 0.3% of chlorobutol have remained stable for more than a year at +4% Repeated freezing and thawing does not affect the activity of batroxobin solutions. Specificity. Batroxobin converts fibrinogen into fibrin by specific cleavage of those Arg-Gly bonds in the a-chain, which leads to release of 4 G. It. Barlow, L. J. Lewis, R. Finlay, D. Martin, and K. Stocker, Thromb. Res. 2, 17 (1973). 5 K. Stoeker and G. H. Barlow in "Defibrinierung mit thrombinghnliehen Sehlangengiftenzymen" (M. Martin and W. Schoop, eds.), pp. 45-63. Huber, Bern, 1974.
222
BLOOD CLOTTING ENZYMES
[18]
fibrinopeptide A, whereas the fl-chain of fibrinogen remains unaffected2 ,7 Prolonged incubation of fibrin with batroxobin causes, just as does t h a t of thrombin, release of the tripeptide Gly-Pro-Arg. 8 Batroxobin neither exerts a direct fibrinolytic activity nor activates plasminogen. Batroxobin acts with a species-dependent preference on fibrinogen of different m a m m a l s " ; hence, coagulation of r a b b i t fibrinogen takes about 10 times longer t h a n the coagulation of h u m a n fibrinogen after additions of equal amounts of batroxobin. This phenomenon might be explained b y structural differences of rabbit and h u m a n fibrinogen as reflected in the sequence analysis of fibrinopeptide A from different mammals. 1° I n contrast to thrombin, batroxobin does not activate h u m a n factor V I I I 11 and neither induces nor affects the platelet's aggregation and release reactions. 12 Therefore, it forms a nonretracting clot with plateletrich h u m a n plasma. ~3 Batroxobin isolated from the venoms of B. asper and B. moojeni, like thrombin, activates factor X I I I , whereas batroxobin from B. marajoensis venom does not activate this factor. 14 Thus, batroxobin from B. asper and B. mo,ojeni venom, in the presence of Ca 2+, forms with prothrombin-free h u m a n plasma, a clot t h a t remains insoluble in 5 M urea or 1% monochloroacetic acid, whereas batroxobin from B. marajoensis forms a clot t h a t is rapidly dissolved in these agents. Batroxobin is able to hydrolyze arginine esters such as N"-benzoyl-L arginine ethyl ester or toluenesulfonyl-L-arginine methyl ester, 15 and it furthermore digests the synthetic fibrinopeptide A analogs B z - P h e - V a l A r g - p N A and d a n s y l - G l y - G l y - V a l - A r g - G l y O M E . 16 Batroxobin originating from the different varieties of Bothrops atrox acts with a different velocity on B z - P h e - V a l - A r g - p N A (Table I I I ) . Activators and Inactivators. Batroxobin-induced coagulation of fibrinogen is accelerated in the presence of imidazole or phenol. Since 6B. Blombiick, M. Blomb~ick, and I. M. Nilsson, Thromb. Diath. Haemorrh. 1, 76 (1957). ' K. Stocker and P. W. Straub, Thromb. Diath. Haemorrh. 24, 248 (1970). SB. ttessel and M. Blomb~ick, FEBS Left. 18, 318 (1971). 9 K. Wik, O. Tangen, and F. McKenzie, Br. ]. Haematol. 23, 37 (1972). lo K. Laki, Fibrinogen 1968, 9 (1968). "S. Lopaciuk and Z. S. Latallo, Abstr. Int. Congr. Thromb. Haematol, $th (1973). 12S., Niewiarowski, J. St. Stewart, N. Nath, A. Tai Sha, and G. E. Liebermann, Am. J. Physiol. in press 18G. DeGaetano, R. Franco, M. B. Donati, A. Bonaccorsi, and S. Garattini, Thromb. Res. 4, 189 (1974). ~ J. McDonagh and R. P. McDonagh, Abstr. Int. Congr. Thromb. Haematol., $th (1973). ,5 j. Soria, C. Soria, J. Yver, and M. Samama, Coagulation 2, 173 (1969). aeK. H. Chen, I. Simpson, J. Bruner-Lorand, and L. Lorand Abstr. Meet. Fed. Am. Soc. Exp. Biol. (1972).
[19]
CROTALASE
223
TABLE III ACTION OF BATROXOBINORIGINATING FROMBothrops marajoensis AND B. moojeni ON Bz-PuE-VAL-ARG-pNA Vm~ (inoles/min) Batroxobin from
K (M)
Per mU
Per BU"
B. marajoensis B. moojeni
1.61 X 10-4 2.86 X 10-4
2 3.9
2.38 X 10-3 0.59 X 10-3
a BU = batroxobin unit. hydrolysis of Bz-Phe-Val-Arg-pNA is also potentiated by imidazole but not by phenol, imidazole m a y be considered as an activator, whereas phenol seems to catalyze fibrin polymerization. Batroxobin is inactivated by 2.5 m M diisopropyl fiuorophosphate, pH 8. In contrast to thrombin, it is not inactivated by incubation for 15 hr at 20 ° in iodoacetamide, 0.1%, pH 7.4, and, whereas thrombin looses its clotting activity after 1 hr of incubation in SDS 0.01%, pH 7, at 20 °, this treatment does not affect the activity of batroxobin. Batroxobin is inactivated neither by thrombin inhibitors, such as heparin, heparinoids, or hirudin, nor by proteinase inhibitors, such as aprotinin, soybean inhibitor, nor by plasmin inhibitors such as e-aminocaproic acid or tranexamic acid. Batroxobin is bound to a2-macroglobulin and looses its clotting activity, whereas its amidolytic activity on the small synthetic substrate Bz-Phe-Val-Arg-pNA remains almost unaffected. 17 1, N. Egberg, Thromb. Res. 4, 35 (1974).
[19] Crotalase B y FRANCIS S. MARKLAND,JR. Crotalase is a thrombinlike enzyme isolated from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). 1 In 1886, Mitchell and Reichert 2 demonstrated that animal blood failed to coagulate after treatment with C. adamanteus venom. They carried out extensive studies on this venom and showed that the globulin (protein) F. S. Markland and P. S. Damus, d. Biol. Chem. 264, 6460 (1971). 2S. W. Mitchell and E. T. Reichert, Smithsonian Contrib. Knowl. 26, 1 (1886).
