Proteolytic destruction of a hemorrhagic toxin from the venom of the habu (Trimeresurus flavoviridis)

The major hemorrhagic principle (HR1B) in the venom of Habu (Trimeresurus flavoviridis), a crotalid, was found to be easily inactivated and hydrolyzed by a commercial protease (Nagarse). The ability of the hydrolysate to produce the toxin-neutralizing antibody has been shown to be comparable to or superior to those of two formol toxoids used as the controls, suggesting that inactivation of HR1B by the protease provides a new method for the preparation of Habu toxoid.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.     

Purification, characterization and substrate specificity of a basic proteinase in the venom of habu (Trimeresurus flavoviridis)

A basic proteinase was purified and characterized from the venom of Habu (Trimeresurus flavoviridis). Its molecular weight, isoelectric point and optimum pH were approx. 24,000, 9.2 and 9, respectively. Susceptibility to several reagents was examined. The proteinase had endopeptidase activity cleaving the Gly-Leu bond in synthetic peptides but no exopeptidase activity. It did not hydrolyze a peptide, Z-Gly-Pro-Leu-Gly-Pro, which had been a good substrate for the major proteinase in the venom. The proteinase cleaved oxidized insulin B chain at five positions: His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Gly23-Phe24 and Phe24-Phe25. From the disappearance of intermediate peptides and the peptides accumulated, the order and the intensity of cleavage of these positions were determined, and the substrate specificity was compared with those hitherto described for hemorrhagic and nonhemorrhagic venom proteinases.     

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.     

Anticoagulant mechanism of factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.     

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.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

The occurrence of HR1b in the venom of the snake Okinawa habu (Protobothrops flavoviridis)

This study describes the first isolation of hemorrhagic metalloproteinase HR1b from the venom of Okinawa habu, and its cDNA cloning. The substrate specificity of isolated HR1b definitely differed from that of HR1a, further supporting the presence of a metalloproteinase distinguishable from HR1a in the venom of Okinawa habu. The deduced amino acid sequence of HR1b showed 99.67% identity with HR1b of Amami habu, with only two amino acid residue replacements.     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.