Physicochemical properties of alpha- and beta-fibrinogenases of Trimeresurus mucrosquamatus venom.

alpha- and beta-Fibrinogenases (EC 3.4.21.5) were purified from Trimeresurus mucrosquamatus venom by the technique of recycling chromatography. Both enzymes were single polypeptide chains and homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation constants of alpha- and beta-fibrinogenases were 2.52 and 3.04 respectively. The molecular weight of alpha-fibrinogenase was 21 500--23 400, and that of beta-fibrinogenase was 25 000--26 000. The contents of proline, glycine and tryptophan were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha- and beta-fibrinogenases were pH 8.1 and 5.7 respectively. The optimal pH of alpha-fibrinogenase was about 7.4 and that of beta-fibrinogenase was around 8.5. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.6, 7.4 and 9.0, while that of beta-fibrinogenase was not significantly affected by the same treatment. Both enzymes showed proteolytic activities toward fibrinogen and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities of the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 17 times that of the crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethane sulfonylfluoride and slightly by tosyl-L-lysine chloromethylketone and cysteine.     

Purification and characterization of a fibrinogenase from the venom of Western Diamondback rattlesnake (Crotalus atrox)

A fibrinogenolytic enzyme was isolated from the venom of Western Diamondback rattlesnake (Crotalus atrox) by a three-step procedure involving gel filtration and anion-exchange chromatography. The molecular weight was estimated as 22 900 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 4.65. The enzyme rapidly destroyed the ability of bovine fibrinogen to form a clot on incubation with thrombin. Incubation of fibrinogen with the fibrinogenolytic enzyme for 5 min resulted in the disappearance of the beta-chain of fibrinogen and the appearance of lower molecular weight fragments. Thus the enzyme can be classified as a beta-fibrinogenase. However, on prolonged incubation of the fibrinogen there was also a partial digestion of the alpha-chain. The fibrinogenase showed no activity towards fibrin or casein or arginine esters. The fibrinogenolytic activity was inhibited by phenylmethanesulphonyl fluoride (PMSF) but was unaffected by EDTA.     

眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究

经Heparin-SepharoseCL-6B亲和层析和SephadexG-150分子筛层析,从中国眼镜蛇毒(Najanajaatra)中纯化出一种可水解纤维蛋白原Aα链的蛋白酶——组分F.SDS-PAGE测得分子量为47100,为一含2%中性己糖的单肽链糖蛋白;它对纤维蛋白、酪蛋白和苯甲酰-L-精氨酸乙酯(BAEE)均无水解作用,也无激活纤溶酶原的作用;100μg皮下或皮内注射,未见出血反应。组分F对酸和热均不稳定。EDTA,EGTA,PMSF和肝素等可抑制其纤维蛋白原溶解活性,benzami-dine,aprotinin和大豆胰蛋白酶抑制因子(SBTI)则无抑制作用。     

A novel alpha-type fibrinogenase from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.     

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.     

Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

Comparative study of four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus (the Chinese habu snake)

Four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus had been isolated and characterized by Sugihara et al. (1980, 1981, 1982, 1983). Immunologically, ME-1, 2, 3 and 4 are identical. The four enzymes hydrolyzed Pro-Phe-Arg-MCA and z-Phe-Arg-MCA. Furthermore, ME-2 slightly hydrolyzed Boc-Val-Pro-Arg-MCA, Boc-Phe-Ser-Arg-MCA and Boc-Ile-Glu-Gly-Arg-MCA. ME-1 cleaved almost simultaneously the Arg(22)-Gly(23) and Phe(25)-Tyr(26) bond of oxidized insulin B chain. ME-2 and 3 also hydrolyzed the same bond of insulin B chain, but the activity was not as potent as ME-1. ME-4 did not cleave the substrate. The four enzymes hydrolyzed C-terminal of arginine in the biologically active peptides. Four arginine ester hydrolases showed fibrinogenolytic activity. ME-1 and 2 first cleaved B beta-chain and then A alpha-chain. On the contrary, ME-3 and 4 cleaved A alpha- and B beta-chain simultaneously. The four enzymes also hydrolyzed fibrinogen in plasma cleaving B beta- and gamma-chain and slightly digesting A alpha-chain. The various inhibitors affected TAME (tosyl-arginine-methylester) and the fibrinogen hydrolytic activity of the four enzymes. All four enzymes had fibrinolytic activity.     

Comparative study of three proteinases from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Three immunochemically distinct proteinases (P-1, 2 and 3) devoid of hemorrhagic activity were isolated from the lyophilized venom of Trimeresurus mucrosquamatus using column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel, CM-Cellulose and Bio-Rex 70. By these procedures, about 7.6, 7.3 and 8.2 mg of purified P-1, 2 and 3 may be obtained from 1 g of crude venom, respectively. The purified proteinases 1-3 were homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. The isoelectric point of P-1 was 8.1; P-2, 9.2; P-3, 9.8. The molecular weights of proteinases 1-3 were determined to be 23,000, 23,500 and 23,000, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, respectively. The purified proteinases 1-3 possessed caseinolytic and fibrinogenolytic activities. These activities were inhibited when the proteinases were incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine, but not with egg white trypsin inhibitor (EWTI) or soybean trypsin inhibitor (SBTI). P-1 cleaved the B beta-chain of fibrinogen first and then the A alpha-chain, whereas P-2 and 3 cleaved the A alpha-chain first and then the B beta-chain. However, these three proteinases did not hydrolyze the gamma-chain.     

眼镜蛇毒中一个弱纤维蛋白原水解活性金属蛋白酶的纯化和性质研究

通过蛋白层析从中华眼镜蛇毒中分离纯化出一个新的纤维蛋白原水解酶atrase A. Atrase A是一个分子量为64.6 kD,等电点为pH 9.6和中性糖含量为4.16%的碱性单链糖蛋白.它具有弱的纤维蛋白原α链水解活性.该活性能被金属螯合剂EDTA, EGTA,1,10 phenanthroline和还原剂DTT完全抑制,而PMSF只能部分抑制该活性,大豆胰蛋白酶抑制剂对其没有影响, 表明atrase A属于金属蛋白酶. Atrase A具有水肿活性和金黄色葡萄球菌抑制活性.它对A549 和K562 细胞没有细胞毒性,但能使贴壁生长的A549细胞解离悬浮. Atrase A没有纤维蛋白、azocasein 、BAEE水解活性,对ADP、胶原诱导的血小板聚集没有明确的抑制作用. 经小鼠皮下注射后没有发现其有出血毒活性.     

Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum

A fibrinolytic enzyme was isolated from a marine green alga, Codium divaricatum, and designated C. divaricatum protease (CDP). This protease effectively hydrolyzed fibrinogen A alpha chain, while it had very low hydrolyzing efficiency for B beta and gamma chains. This property was similar to that of alpha-fibrinogenase isolated from snake venom. Protease activity peaked at pH 9, and was completely inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), identifying it as a serine protease. Its molecular form was single polypeptide structure and molecular weight was estimated as 31,000 by SDS-PAGE. Fibrinogen clotting enzyme was also identified in a fraction by ion-exchange chromatography. Analysis of clots formed by the enzyme and by thrombin by SDS-PAGE showed that the fibrinogen clotting enzyme would act like thrombin and have high substrate specificity.     

Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom

Snake venoms of the Viperidae family contain a numberof proteins that cause hemostatic disturbances. Enveno-mation of this family is characterized by hemorrhage,edema, local tissue damage, myonecrosis, fibrinolytic andkinin releasing activities [1]. In southeastern Brazil, theviper Bothrops jararaca (Viperidae) is responsible for 90%of snakebite accidents [2]. The enzymes that have proteolytic, coagulate andhemorraghic activities can activate or interfere withthe process of coagulation, and…     

Cardiovascular alterations induced by the venom of Lachesis muta (serpentes: Viperidae) and its fibrinogenolitic enzyme

The fibrinogenolytic activity of Lachesis muta stenophyrs venom was studied. Wistar rats catheterized at carotid artery and jugular vein were inoculated with crude venom or enzyme and changes in arterial pressure, cardiac frequency and electrocardiogram were monitored. The enzyme induced a greater fibrinogen reduction than the crude venom without any cardiovascular or histological alteration. In vitro crude venom coagulated blood whereas the enzyme reduced fibrinogen in 23%. Results suggest the potential use of the fibrinogenolytic enzyme as antithrombotic agent.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Fibrinogenolytic toxin from Indian monocled cobra (Naja kaouthia) venom

A fibrinogenolytic toxin of molecular weight 6.5 kDa has been purified from the venom of Indian monocled cobra (Naja kaouthia) by repeated cation exchange chromatography on CM-sephadex C-50. The purified toxin did not show any phospholipase activity but was mildly hemolytic on human erythrocytes. This toxin, called Lahirin, cleaved fibrinogen in a dose- and time-dependent manner. The digestion process apparently started with the A alpha chain, and gradually other lower-molecular-weight chains were also cleaved to low-molecular-weight peptides. The fibrinolytic activity was completely lost after treatment with ethylene di-amine tetra acetic acid (EDTA). However, exposure to 100 degree C for 1 min or pre-treatment with phenyl methyl sulfonyl fluoride (PMSF) did not affect the fibrinolytic activity. Cleavage of di-sulphide bonds by beta-mercaptoethanol or unfolding the protein with 4 M urea caused complete loss of activity of pure Lahirin.     

Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.     

Purification and properties of the main coagulant and anticoagulant principles of Vipera russellii snake venom

Vipera russellii venom was separated into thirteen fractions by means of DEAE-Sephadex A-50 column chromatography. Fraction III possessed anticoagulant and phospholipase A activities and Fraction XI possessed procoagulant and caseinolytic activities, both were further purified by gel filtration on Sephacryl S-200 column. Purified procoagulant (Component II) was a two-chain protein with molecular weight of 86 000 consisting of A-chain (Mr 66 000) and B-chain (Mr 20 000). It was a glycoprotein containing 7.8% neutral sugar and 715 amino-acid residues. The procoagulant activity was 10-times that of the crude venom. It was an acidic proteinase with isoelectric point of pH 4.2. Upon heat treatment at 60 degrees C, Component II was stable at pH 5.5 and 7.2 for 3 h, but was destroyed completely after 30 min at pH 8.9. It was devoid of esterase or amidase activity. Purified anticoagulant (Component I) was a single peptide chain with molecular weight of 16 000. It was carbohydrate free and contained 136 amino-acid residues. It was a basic protein with an isoelectric point of larger than pH 10. It was a potent phospholipase A with an enzymatic activity of 510 +/- 30 mumol/min per mg using phosphatidylcholine as substrate, and 1 microgram/ml was sufficient to cause 100% hemolysis by the indirect hemolytic method. Upon heat treatment at 90 degrees C, Component I was heat stable at pH 5.5 for more than 3 h, but was destroyed completely after 2 h at pH 7.2 and 8.9. The anticoagulant activity of Component I could be neutralized by platelet factor 3, tissue thromboplastin and cephalin.     

Cleavage of the A alpha-chain of fibrinogen and the alpha-polymer of fibrin by the venom of spitting cobra (Naja nigricollis)

The effect of Naja nigricollis venom of fibrinogen and highly crosslinked fibrin was examined by SDS-polyacrylamide gel electrophoresis of the reduced products of venom treatment. The venom contains a proteolytic activity which degraded the A alpha-chain of fibrinogen, but had no apparent effect on the B beta- or gamma-chains of the molecule. The venom also readily degraded the alpha-polymer or highly crosslinked fibrin, without apparent cleavage of the beta-chain or the gamma-dimer of fibrin. The venom had no observed effect on plasminogen, indicating that the effects on the A alpha-chain and the alpha-polymer are by direct action of the venom, and not due to activation of plasminogen. The fibrinogenolysis was inhibited by EDTA or 1,10-phenanthroline. Inhibition with EDTA could be reversed by the addition of Zn2+. The fibrinogenolysis was optimal between pH 7 and 8, consistent with the expected pH optimum for a Zn2+ metalloproteinase.     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.