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 μg, respectively. These purified hemorrhagic factors were not lethal at 15 μg/g in mice. Factor a hydrolyzed the Bβ chain of fibrinogen, while factor b hydrolyzed the Aα 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.     

Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus.     

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.     

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.     

Primary structures of cytotoxic factors isolated from habu (Trimeresurus flavoviridis) venom

The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.     

A potent platelet aggregation inducer purified from Trimeresurus mucrosquamatus snake venom

A non-coagulant platelet aggregation inducer (called platelet 'aggregoserpentin') was isolated from Trimeresurus mucrosquamatus snake venom by CM-Sephadex chromatography and purified by gel filtration. It was homogeneous as judged by the ultracentrifugal analysis and electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weight was estimated to be 68 000 as judged by the SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75. The ultracentrifugal analysis gave 3.19 Svedberg units. It was a protein-polysaccharide complex containing 340 amino acid residues and 50% carbohydrate per molecule. The isoelectric point was pH 5.4. It did not possess any of the hydrolase enzymatic properties which were found in the crude venom. The minimal concentration of 'aggregoserpentin' necessary to induce platelet aggregation was 10 ng/ml, about one four-hundredth of that of the crude venom. It did not cause lysis of platelets because lactate dehydrogenase was not found in supernatant after complete aggregation. An intravenous injection of 'aggregoserpentin' (35 microgram/kg) into rabbit ear marginal vein caused marked decrease of platelet number to approx. 10-20% of that of the control.     

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.     

Isolation and characterization of hemorrhagic toxin from the venom of Trimeresurus elegans

1. Hemorrhagic toxin from the venom of Trimeresurus elegans was purified in a homogeneous form using gel filtration and ion exchange chromatographies. 2. Hemorrhagic toxin possessed hemorrhagic and TAME (tosyl-L-arginine methyl ester) hydrolytic activities. These activities were inhibited when hemorrhagic toxin was incubated with benzamidine or N-bromosuccinimide. 3. Its mol. wt was 28,500 and the isoelectric point was 7.4. 4. This toxin contains ca 1.5 mol Ca per mol of protein.     

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.     

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.     

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.     

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.     

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.     

Isolation and characterization of nerve growth factor from Vipera lebetina (snake) venom

Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9-10.5. All isoforms have identical molecular weights of 32,500.     

Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of 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 phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.     

湖南烙铁头蛇毒中一个新的舒缓激肽增强肽（TmF）

通过 70 %冷甲醇抽提、SephadexG 15分子筛和反相高效液相色谱C1 8层析 ,从湖南产烙铁头蛇毒 (Trimeresurusmucrosquamatus)冻干粉中纯化得到一个新的舒缓激肽增强肽 (BPP) ,命名为TmF。该小肽的氨基酸序列为pGlu Gly Arg Pro Leu Gly Pro Pro Ile Pro Pro (pGlu表示焦谷氨酸 )。序列结果分析表明 ,TmF和已经分离得到的BPPs有很高的序列同源性。MSI MS测定其分子量为 1.110 7kD。TmF的生物学活性和药理学活性检测的结果表明 ,它增强舒缓激肽 (BK) ( 1mg L)诱导的离体豚鼠回肠纵行肌收缩的活性为 ( 1.13± 0 .3)单位 (mg L) ;TmF ( 5 .0× 10 - 4mg kg)可以增强约 ( 14± 2 )mmHg的由BK( 5 .0× 10 - 5mg kg)诱导的舒张压下降 ;在抑制剂试验中 ,不同剂量的TmF和 5× 10 - 2 mg的血管紧张素转化酶保温 30min ,结果表明大约2 .0× 10 - 3mg的TmF表现出对ACE水解活性的半数抑制率 (IC50 )。     

Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom

A protein isolated from timber rattlesnake (Crotalus horridus horridus) venom by ion-exchange and high-pressure liquid chromatography is hemorrhage inducing and lethal to mice (LD50 of 10 micrograms/g of body weight). It is a Ca2+- and Zn2+-containing proteinase and has the ability to hydrolyze hide powder azure. Atomic absorption spectroscopy shows 2.5 Ca2+ and 1 Zn2+ per protein monomer. The proteinase activity is destroyed by incubation with disulfide-reducing agents and by dialysis against ethylenediaminetetraacetate. Coincident with the loss of proteinase activity is a corresponding loss of lethal and hemorrhagic activities, suggesting that all three are related. Attempts to replace the metals and restore activity have been unsuccessful. Amino acid analysis and isoelectric focusing reveal that this component is an acidic protein (pI = 5.1) containing about 20 disulfide bonds and 507 residues. Reduction of one disulfide bond per molecule decreases proteinase activity by 50% while reduction of eight disulfide bonds decreases activity by 80%. Loss of hemorrhagic activity parallels the decrease in proteinase activity.