Marina T. Assakura

Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (Jararaca).

Bothropasin, one of the proteases from the venom of Bothrops jararaca active on casein, was isolated by ammonium sulfate precipitation, DEAE-cellulose and DEAE-Sephadex A-50 chromatographies and Sephadex G-100 column filtration. The preparation possessed no other detectable activities which are present in the crude venom. Addition of Ca2+ during purification stabilized the enzyme. The endopeptidase was inhibited by EDTA and EGTA; Ca2+ did not restore the activity of the inhibited enzyme. The material was homogeneous by polyacrylamide gel electrophoreses at different pH values, immunoprecipitation and crossed immunoelectrophoresis. By SDS-polyacrylamide gel electrophoresis the denatured and reduced enzyme had only a 48,000 molecular weight band. In the presence of 6 M guanidine-HCl and 0.1 M beta-mercaptoethanol the preparation showed a value of 49,870 by sedimentation equilibrium. The native tertiary structure of the protein is dependent on S-S and metal bonds. The denatured and reduced enzyme, in the presence of EDTA, showed a molecular weight of 37,300 by sedimentation equilibrium, a value which was also confirmed in SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed five peptide bonds: His-Leu (5-6), His-Leu(10-11), Ala-Leu(14-15), Tyr-Leu(16-17) and Phe-Phe(24-25) in the B-chain of oxidized insulin.

Comparison of immunological, biochemical and biophysical properties of three hemorrhagic factors isolated from the venom of Bothrops jararaca (jararaca)

Compared to the crude velonom of Bothrops jararaca, which needs 5000 ng to produce a hemorrhagic spot of 1 cm2 on rabbit skin, the isolated hemorrhagic factors HF1, HF2 and HF3 require 100, 20 and 15 ng of protein, respectively. Although these hemorrhagic factors possess different biochemical and biophysical properties, they are immunologically related proteins. The hemorrhagic, as well as the proteolytic, activities of these factors are destroyed by EDTA, acidic pH or heat treatments.

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (jararaca pintada)

Two hemorrhagic factors were isolated from the venom of Bothrops neuwiedi (jararaca pintada) by ammonium sulfate precipitation followed by chromatography on DEAE-Sephadex A-50 and DEAE-cellulose DE-32, gel filtration on Sephadex G-100 and polyacrylamide-gel electrophoresis. These factors were named neuwiedi hemorrhagic factors NHFa and NHFb. They are acidic proteins of pI 4.2-4.3 and consist of single polypeptide chains of molecular weights 46,000 and 58,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The hemorrhagic activity of NHFb is 23 times stronger than that of NHFa. Both hydrolyse casein, although NHFa is about 20 times more active than NHFb. They are metalloproteins inhibited by EDTA and 1,10-phenanthroline. NHFa and NHFb are serologically closely related antigens. These two factors are recognized as identical antigens by horse serum against crude Bothrops neuwiedi venom. However, the rabbit specific antiserum was able to differentiate NHFa from NHFb showing, nevertheless, that they have common determinants apart from specific determinants for each one.

Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca)

Moojeni protease A was purified from the venom of Bothrops moojeni by chromatography on Sephadex G-100, DEAE Sephadex A-50 and rechromatography on Sephadex G-100. The enzyme shows one protein band in polyacrylamide gel electrophoresis at pH 8.5 or at pH 4.3. The pI of moojeni protease A was approximately 7.7. In immunoelectrophoresis it migrates to the cathode. The enzyme was homogeneous by polyacrylamide gel electrophoresis, immunoelectrophoresis and analyses in the ultracentrifuge. The S20,w and D20,w are 2.68 S and 10.34 X 10(-7) cm2/sec, respectively. The molecular weight calculated by s/D ratio was 22,500 and a value of 22,800 was obtained by sedimentation equilibrium. In SDS-polyacrylamide gel electrophoresis the enzyme exhibits a single polypeptide chain of approximately 20,400 mol. wt under denaturating conditions. In water or low salt solution it undergoes denaturation and autolysis. The enzyme is also unstable at acidic pH and to heat treatment and precipitates in the presence of metal chelating compounds such as EDTA or 1,10 phenanthroline. Leucine, the NH2-terminal amino acid of moojeni protease A is blocked after EDTA treatment. The proteolytic activity of this enzyme increases about 20% in the presence of Ca2+; Mg2+ has no effect and other divalent cations cause inhibition. The removal of Ca2+ ions by oxalate causes about 20% inhibition; the activity was restored by addition of Ca2+.

Hemorrhagic, fibrinogenolytic and edema-forming activities of the venom of the colubrid snake Philodryas olfersii (green snake)

The venom of P. olfersii has high hemorrhagic, edema-inducing and fibrin(ogen)olytic activities. It is devoid of thrombin-like, procoagulant, phospholipase A2 and platelet aggregating enzymes. The main activities are metalloproteinases inhibited by metal chelators (EDTA and 1,10-phenanthroline) and sulfhydryl compounds (DTT and cysteine). The hemorrhagic and fibrinogenolytic enzymes were partially purified by gel filtration on HPLC. The hemorrhagic activity of the venom was neutralized by commercial horse antivenoms to Bothrops species, as well as by rabbit antisera specific for hemorrhagic factors isolated from these Bothrops venoms. No immunoprecipitin reactions were obtained, indicating that the few epitopes of the P. olfersii hemorrhagin are involved in these neutralization reactions. The fibrinogenolytic enzyme cleaves A alpha-chain more quickly than the B beta-chain of human fibrinogen. The venom also solubilizes fibrin. This solubilization appears to occur from the hydrolysis of unpolymerized alpha-chain and cross-linked gamma-gamma dimer. The fibrin peptide products are distinct from those produced by plasmin.

Isolation and characterization of five fibrin(ogen)olytic enzymes from the venom of Philodryas olfersii (green snake)

Five distinct fibrin(ogen)olytic proteinases PofibC1, C2, C3, H and S were isolated by gel filtration and ion-exchange chromatographies. PofibC1, C2, C3 and H are metalloproteinases inhibited by ethylenediamine tetracetic acid (EDTA) or 1,10-phenanthroline. Only PofibH had hemorrhagic activity. PofibS is a serine proteinase, inhibited by phenylmethylsulfonyl fluoride (PMSF) or Torresea cearensis trypsin inhibitor (TCTI). All five enzymes were inhibited by dithiothreitol (DTT) or dithioerythritol (DTE). PofibC1 and C2 presented the same mol. wt of 47,000 and are acidic proteins of pI 6.2 PofibC3 is a basic proteinase of pI 8.5 and mol. wt 45,000. The hemorrhagic proteinase PofibH had a mol. wt of 58,000 and pI of 4.6 and PofibS had a mol. wt of 36,000 and pI of 4.5. The five proteinases degraded fibrin and fibrinogen. PofibC1, C2, C3 and H degraded preferentially A alpha-chains while PofibS cleaved concomitantly A alpha and B beta-chains of fibrinogen. None of these enzymes cleaved the gamma-chain of fibrinogen. When correlated with the thrombin delay time, the most active was PofibS, while PofibH and PofibC1 showed almost no activity. The proteinases also differed in the peptide cleavage of B-chain of insulin. Philodryas olfersii venom promoted in vivo a loss of the circulant plasma fibrinogen, as was observed in experiments with rats.

Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.

Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.

Pathological changes in muscle caused by haemorrhagic and proteolytic factors from Bothrops jararaca snake venom

Haemorrhagic factor HF2 and bothropasin, two metalloproteins isolated from the venom of Bothrops jararaca, caused haemorrhage followed by myonecrosis and arterial necrosis after i.m. injection in mice. The effects of HF2 were qualitatively similar to those of bothropasin and crude B. jararaca venom, but its potency was about 20 times higher. The haemorrhagic and necrotizing actions of these components are unrelated to their proteolytic activity on casein.

Antigenic relationship of hemorrhagic factors and proteases isolated from the venoms of three species of Bothrops snakes.

By comparative studies of the immunological properties of the metalloproteins (hemorrhagic factors and proteases) isolated from the venoms of Bothrops jaracaca, Bothrops neuwiedi and Bothrops moojeni, it was found that the hemorrhagic factors contain common antigenic determinants and the proteases were immunologically distinct entities. The rabbit antisera raised for the hemorrhagic factors not only neutralized the hemorrhagic activities of the respective factors but also activities of the other hemorrhagic factors. Although the homology among these proteins are not yet known, these studies have shown that the hemorrhagic factors must have a similar partial structure which includes the catalytic hemorrhagic active site.