A hemorrhagin as a metalloprotease in the venom of Trimeresurus purpureomaculatus: purification and characterization.

A major hemorrhagin was purified from the venom of the Thai green pit viper (Trimeresurus purpureomaculatus) by gel filtration, ion-exchange and affinity chromatography. A 15-fold purification was achieved with an overall yield of 7% of hemorrhagic activity. The hemorrhagin was homogeneous according to disc- and SDS-PAGE as well as on immunodiffusion. The molecular weight determined by SDS-PAGE was 72kDa. The purified hemorrhagin expresses proteolytic activity with heat-denatured casein and hide powder azure, but it was free of AE-hydrolase and phospholipase activities. Both hemorrhagic and proteolytic activities were inhibited by EDTA, suggesting that the hemorrhagin is a metalloprotease. The hemorrhagin hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen, whereas it hydrolyzed only type IV native collagen. The hemorrhagic activity was neutralized by Thai green pit viper antivenom raised to Trimeresurus albolabris venom.

Isolation of the major lethal toxin in the venom of Bungarus flaviceps.

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments.

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.

The antihemorrhagic factor, erinacin, from the European hedgehog (Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains.

From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, alpha and beta, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine-HCl, erinacin dissociates into alpha-subunits and beta-subunit decamers. From these results the subunit assembling of erinacin has been formulated as alpha(10).2beta(10). The molecular weight of the subunits and of the beta-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.

Development of reversed passive latex agglutination for detection of Thai cobra (Naja kaouthia) venom.

A simple, rapid, and sensitive diagnostic kit for detecting Thai cobra (Naja kaouthia) venom was developed using latex particles sensitized with venom specific immunoglobulin. The kit is capable of detecting 25-50 ng/ml of Thai cobra venom. The capability was not affected by human plasma. Specificity of the kit was proven using snake venoms from Vipera russelli, Calloselasma rhodostoma, Trimeresurus albolabris, Naja siamensis, Ophiophagus hannah, and Bungarus fasciatus. The diagnostic kit does not lose its capability under refrigeration for two months and by lyophilization.

Genus specific neutralization of Bungarus snake venoms by Thai Red Cross banded krait antivenom.

Thai commercial antivenom raised to Bungarus fasciatus venom neutralized the lethal activity of all Thai Bungarus venoms tested in in vitro neutralization experiments. The neutralizing capacities against B. fasciatus and B. candidus venoms were almost the same, but that against B. flaviceps venom was significantly greater. The efficacy of the antivenom was confirmed in in vivo neutralization experiments also. Results of immunochemical analyses supported results of the animal experiments suggesting the presence of genus specific neutralization.

Envenomation of mice by Thai cobra (Naja kaouthia) venom: tolerable venom concentration and exposure time.

Naja kaouthia venom appeared in circulation rapidly after intramuscular injection into mice. The venom concentration attained a maximum level with all doses examined after 20 min. The half value of the maximum level was obtained 1 min after injection when a dose of 4LD50 was used. A critical venom concentration endangering mice was assessed from venom concentration in the sera of mice envenomed with sublethal dose (LD50). A fatal condition was produced within 30 min at a venom concentration of 200-300 ng/ml or within 50 min at a venom concentration of 100-150 ng/ml.

Efficacy and cross reactivity of Thai green pit viper antivenom among venoms of Trimeresurus species in Thailand and Japan.

The efficacy of the Thai green pit viper antivenom to neutralize lethal, hemorrhagic, and enzyme activities of Trimeresurus venoms was examined using venoms of Trimeresurus albolabris, T. macrops, and T. flavoviridis (Japanese Habu). Antivenom against Japanese Habu venom was also used to study immunological cross reactivity among Trimeresurus venoms. Thai green pit viper antivenom was comparably effective to Habu antivenom to neutralize all activities. Distinct cross neutralization was demonstrated indicating the presence of genus specific protection by Thai and Japanese Trimeresurus antivenoms. Results of immunoblotting analyses indicated that Thai and Japanese Trimeresurus venoms contain many cross-reactive protein components.

Comparison of antihemorrhagic activities in skeletal muscle extracts from various animals against Bothrops jararaca snake venom.

Antihemorrhagic activities of skeletal muscle extracts from various animals were compared in inhibiting the hemorrhagic activity of Bothrops jararaca venom. The muscle extracts of the European hedgehog (Erinaceus europaeus) exhibited the strongest activity, followed by those of other insectivores such as the shrew (Crocidura russula) and mole (Talpa europaea). The antihemorrhagic activities of muscle extracts from experimental animals such as mice, rats, guinea-pigs, hamsters and rabbits were negligible.

Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes.

Specificity of two different types of phospholipase A2 (PLA2) inhibitory proteins from the blood plasma of venomous snakes was investigated. Two Crotalidae inhibitors, having a carbohydrate recognition domain (CRD) in their sequences, inhibited specifically the group-II acidic PLA2s of their own snake venom. On the other hand, Elapidae inhibitor, having two tandem patterns of cysteine residues found in proteins of the Ly-6 superfamily, inhibited not only the group-I PLA2 from its own snake venom but also the group-I, -II, and -III PLA2s from other snake venom. Amino acid sequences of PLA2s that were specifically inhibited by the inhibitors were compared with those of the other PLA2s. A unique aromatic patch structure appeared on the group-II acidic PLA2s was suggested to be involved in the binding to the Crotalidae inhibitors; and residues located in or close to the Ca2+ binding loop of PLA2, in the binding to the Elapidae inhibitor.

A protease from the marine sponge Callyspongia schulzi.

Aqueous extracts of 25 marine sponge species (from coral reefs of Papua New Guinea) were screened for proteolytic activity. Only one sponge, Callyspongia schulzi, showed remarkable activity. A protease hydrolyzing casein as well as the synthetic substrate alpha-N-benzoyl-L-arginine ethyl ester was isolated from the sponge extract by gel filtration, ion-exchange and HPLC absorption chromatography. The enzyme was homogenous in SDS-PAGE exhibiting an apparent molecular weight of 80 kDa. Its pH optimum was in the range of 9-11, it was remarkably heat-stable and was not inhibited by phenylmethane sulfonylfluoride, soybean trypsin inhibitor, aprotinin or alpha1-antitrypsin, but by EDTA and 1,10-phenanthroline suggesting properties of a metalloprotease. The protease hydrolyzed the oxidized insulin B-chain between Arg22-Gly23 and Lys29-Ala30, similar to trypsin.

Cross-neutralization of Thai cobra (Naja kaouthia) and spitting cobra (Naja siamensis) venoms by Thai cobra antivenom.

The neutralizing capacity of antivenom prepared against Thai cobra (Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra (Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 LD50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.

Erinacin, an antihaemorrhagic factor from the European hedgehog, Erinaceus europaeus.

An antihaemorrhagic factor named erinacin was purified from the skeletal muscle extract of the European hedgehog, Erinaceus europaeus, by ammonium sulfate precipitation followed by various steps of ion-exchange (DEAE-cellulose), absorption chromatography (hydroxylapatite), and gel filtration (cellofine gel). A 625-fold purification was achieved with an overall yield of 19% antihaemorrhagic activity. The protein effectively inhibited the activity of Bothrops jararaca venom haemorrhagin and did not inhibit the enzymatic activity of trypsin and chymotrypsin. Erinacin is a large molecule (about 1,000,000 mol. wt). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two subunits: one with an apparent mol. wt of 35,000 forming a larger subunit (350,000) by cross-linking with disulfide bridges, and a second with a mol. wt of 39,000 without disulfides. Dissociation of erinacin into its subunits resulted in complete loss of its antihaemorrhagic activity.

Crystal structure of H2-proteinase from the venom of Trimeresurus flavoviridis.

The crystal structure of the zinc-protease, H2-proteinase, isolated from the venom of Trimeresurus flavoviridis has been determined. The crystallographic R factor is 0.176 for 10,635 reflections with Fobs > 2sigma(Fobs) in the 8.0 to 2.2 Angstrom resolution range. The enzyme has two domains with a cleft in which a catalytic zinc atom is located. The N-terminal domain is composed of four helices around a central five-stranded beta-sheet. The irregularly folded C-terminal domain contains one helix and two disulfide bridges. These two domains are linked by a disulfide bridge. In the zinc environment, the catalytic zinc atom forms a distorted tetrahedral coordination with three histidines and one catalytic water molecule, and the methionine-containing turn is structurally conserved. These are distinctive features of the metzincins, one of the zinc metalloprotease superfamilies. The entire structure shows good agreement with that of two Crotalus snake venom proteases, adamalysin II and atrolysin C. The H2-proteinase, however, contains no structural calcium ions, and differences of disulfide configurations and the coordination of the catalytic water molecule exist as compared with the other two proteases.

Crystallization and preliminary X-ray study of H2-proteinase from the venom of Trimeresurus flavoviridis.

H2-proteinase, a non-hemorrhagic metalloproteinase from the venom of Trimeresurus flavoviridis, has been crystallized by vapor diffusion from solutions containing ammonium sulfate. The crystals belong to the tetragonal space group, P41212 or P43212, with unit cell dimensions of a = b = 77.8 A and c = 82.3 A. The asymmetric unit contains one protein molecule. Diffraction data for a native crystal were collected up to 2.0 A resolution.

Hemorrhagic principles in the venom of Bitis arietans, a viperous snake. II. Enzymatic properties with special reference to substrate specificity.

The optimal pH of the proteinase activity of hemorrhagins, BHRa and BHRb, isolated from the venom of Bitis arietans (puff adder) is pH 9. The activity was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline and 8-hydroxyquinoline, but not by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, suggesting that they are metalloproteinases. The hemorrhagins hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen. On the other hand, only type IV native collagen was hydrolyzed. Gel electrophoretic profiles of type IV collagen hydrolysates suggested that the hemorrhagins affect the collagen helical chains at different cleavage sites. The hemorrhagins hydrolyzed several synthetic peptides such as angiotensin I and luteinizing hormone-releasing hormone, but not synthetic substrates for bacterial and animal collagenases. The hydrolysis of various peptides indicated that the hemorrhagins are endopeptidases. The insulin B chain is cleaved by BHRa and BHRb at 11 and 10 positions, respectively. The substrate specificity of the hemorrhagins was compared with those of known hemorrhagic and nonhemorrhagic venom proteinases.

Hemorrhagic principles in the venom of Bitis arietans, a viperous snake. I. Purification and characterization.

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbreviated as BHRa and BHRb) were purified from the venom of the viperous snake Bitis arietans (puff adder) by gel filtration, ion-exchange and absorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic activity. The hemorrhagins were homogeneous according to disc- and SDS-polyacrylamide gel electrophoresis and immunodiffusion. BHRa and BHRb consist of 623 and 685 amino-acid residues and their apparent molecular weights were 68,000 and 75,000, respectively. They were also immunologically distinct. The purified hemorrhagins express proteolytic activity with heat-denatured casein and hide powder azure. The proteolytic activity with heat-denatured casein was almost the same as that of the crude venom, but that with hide powder azure was less than one-tenth of that of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A2 activities and they are acid labile hemorrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cysteine and by polyvalent anti-snake serum, but not by phenylmethanesulfonyl fluoride or soybean trypsin inhibitor.

Muscle extract of hedgehog, Erinaceus europaeus, inhibits hemorrhagic activity of snake venoms.

The antihemorrhagic activity of muscle extract of hedgehog, Erinaceus europaeus, was tested on various snake venoms with hemorrhagic activity. The extract inhibited strongly hemorrhagic activity of venoms from Bitis arietans, Bothrops jararaca and Vipera latastei gaditana, and remarkably that of venoms from Agkistrodon halys blomhoffi, Bitis gabonica rhinoceros, Bitis nasicornis, Bothrops atrox asper, Crotalus horridus horridus and Vipera berus. The antihemorrhagic activity against eight other snake venoms was below the detection level.

Role of Ca2+ in the binding of phospholipase A2 with a monomeric substrate and with its amide-type analog.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by Group I phospholipases A2 (PLA2s) from Pseudechis australis, Naja naja atra, and bovine pancreas and by Group II enzymes from Vipera russelli russelli, Agkistrodon halys blomhoffii, and Trimeresurus flavoviridis, were studied by the pH-stat assay method at 25 degrees C, pH 7.5-8.2, and an ionic strength of 0.1 or 0.2 in the absence or presence of an amide-type substrate analog, 2-dodecanoyl-amino-1-hexanol-phosphoglycol. The binding of genuine substrate to the Group II enzymes and that of its analog to the Groups I and II enzymes were markedly facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of genuine substrate to the Group I enzymes was found to be independent of the Ca2+ binding. The former result suggests that the structures of the Group II enzyme-genuine substrate complexes and both types of enzyme-analog complexes are generally stabilized by the Ca2+ binding, whereas the latter indicates that the structures of the Group I enzyme-genuine substrate complexes are already similar to those of their Ca2+ complexes and that, therefore, these enzyme-substrate interactions are independent of the Ca2+ binding.