Characterization of a hemorrhagic factor, LHF-I, isolated from the bushmaster snake (Lachesis muta muta) venom.

Hemorrhagic factor I (LHF-I) was previously purified from the venom of the bushmaster snake (Lachesis muta muta). In terms of biochemical and immunological properties, LHF-I is a glycoprotein (mol. wt 100,000, pI 4.7) consisting of two subunits; it loses its activity following mercaptoethanol treatment. LHF-I contains 0.7 g-atom zinc and 1.2 g-atom calcium per mole protein. The hemorrhagic and the proteinase activities are inhibited by EDTA; subsequent addition of Ca2+ or Mg2+ does not reverse the EDTA-induced inhibition of the hemorrhagic activity. The metalloenzyme does not hyrolyze arginine esters and is devoid of phospholipase A2 activity. It hydrolyzes the A alpha- > B beta-chain of fibrinogen without clot formation and hydrolyzes selectively the alpha-chain of fibrin, leaving the B beta- and tau-chains unaffected. Antibodies to the hemorrhagic factor in bushmaster venom were produced by immunizing rabbits with the purified protein. The antibody was purified by protein-A affinity chromatography. This antibody was also used to screen other Crotalinae venom samples for immunologically similar epitopes by ELISA assay. The purified antibody reacted only with LHF-I and two samples of bushmaster venom from different geographical locations.

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.

Hydrolytic specificity of three basic proteinases isolated from the venom of Bothrops moojeni for the B-chain of oxidized insulin.

The hydrolytic activity of three basic proteinases isolated from Bothrops moojeni venom was determined on the B-chain of oxidized insulin. The serine proteinases MSP1 and MSP2 cleave the insulin B-chain at identical positions and in the same order of bond cleavage. Cleavage occurs first at the Arg-Gly(22-23) position, followed by hydrolysis of the Lys-Ala(29-30) peptide bond. The metalloproteinase MPB differs from the serine proteinases in cleaving the insulin B-chain very rapidly at four positions: Ser-His(9-10), Ala-Leu(14-15), Tyr-Leu(16-17) and Phe-Phe(24-25).

Basic proteinases from Bothrops moojeni (caissaca) venom--I. Isolation and activity of two serine proteinases, MSP 1 and MSP 2, on synthetic substrates and on platelet aggregation.

Two serine proteinases, MSP 1 and MSP 2, were isolated from Bothrops moojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoproteins. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 presented two close protein bands corresponding to the mol. wts of 34,000 and 32,500. MSP 2 behaved as a single-chain protein with a mol. wt of 38,000. Specific esterolytic activities of MSP 1 and MSP 2 on alpha-N-tosyl-L-arginine methyl ester (TAME) are 33 mumol min-1 mg-1 and 184 mumol min-1 mg-1, respectively. The most sensitive substrates for the amidolytic activity of both proteinases were the thrombin substrate D-Phe-pipecolyl(Pip)-Arg-4-nitroanilide(Nan) and the glandular kallikrein substrate D-Val-Leu-Arg-Nan. MSP 1, in a concentration of 10(-8) M, causes platelet aggregation in platelet-rich plasma and washed platelets. It also enhances the ADP-induced platelet aggregation. Prostaglandin E1 (PGE1), phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA) abolished completely the aggregation induced by MSP 1. Torresea cearensis trypsin inhibitor (TCTI) inhibited both amidolytic (Ki = 1.96 x 10(-7) M) and platelet-aggregating (Ki = 1.66 x 10(-7) M) activities of MSP 1. The esterolytic activity of MSP 1 and MSP 2 was completely abolished by PMSF, only partially by soybean trypsin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI (Ki = 0.7 x 10(-7) M).

Basic proteinases from Bothrops moojeni (caissaca) venom--II. Isolation of the metalloproteinase MPB. Comparison of the proteolytic activity on natural substrates by MPB, MSP 1 and MSP 2.

A basic metalloproteinase active on casein was isolated from Bothrops moojeni venom by chromatography on Sephadex G-100, DEAE-Sephacel, SP-Sephadex C-50 and Sepharose 12. The enzyme, named MPB, is not hemorrhagic and presents only traces of blood-clotting activity. On polyacrylamide gel electrophoresis at pH 4.3, MPB presented a single and diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme presented two protein bands corresponding to mol. wts of 65,000 and 55,000, which stained with Schiff's reagent. The proteolytic activity of MPB was inhibited by ethylenediaminetetracetate, 1,10-phenanthroline and dithiothreitol. The proteolytic activity of MPB and the serine proteinases MSP 1 and MSP 2 on natural substrates indicates differences in hydrolytic specificity among these enzymes. All fibrinogen chains were degraded by the three proteinases, but MPB is the most active. On fibrin, the proteinases hydrolyzed only the alpha-chain and alpha-polymer, leaving the beta-chain and gamma-dimer apparently untouched. The native type I collagen was partially hydrolyzed by the three enzymes but no digestion product was detected. On the contrary, calf and guinea-pig skin type I gelatins were readily digested by MSP 1 and MSP 2 producing different hydrolysis patterns. MPB was the least active proteinase on the gelatins. The digestion of fibronectin showed an inversion in the specificity of these proteinases. MPB was the most active on fibronectin, while MSP 1 and MSP 2 promoted a faint, partial hydrolysis on this protein.

Proteolytic specificity of moojeni protease A isolated from the venom of Bothrops moojeni.

Moojeni protease A, a proteolytic enzyme isolated from Bothrops moojeni venom, hydrolyzes type I collagen, gelatin, fibrinogen, fibrin and the B-chain of oxidized insulin. The proteinase cleaves the A alpha-chain faster than the B beta-chain of human fibrinogen and shows no effect on the gamma-chain. Fibrin solubilization appears to occur from the hydrolysis of the alpha-polymer and unpolymerized alpha-chain. The enzyme cleaves the Ala(14)-Leu(15) bond of the oxidized insulin B-chain most rapidly, followed by splitting the Ser(9)-His(10) bond. The Tyr(16)-Leu(17) and Gly(20)-Glu(21) cleavage sites were hydrolyzed slightly more slowly, while the peptide bonds His(5)-Leu(6), His(10)-Leu(11), Glu(21)-Arg(22), Gly(23)-Phe(24) and Phe(24)-Phe(25) were more resistant to the enzyme attack. Small synthetic peptides were not hydrolyzed by moojeni protease A.

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.

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta).

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.

Purification of a proteinase inhibitor from the plasma of Bothrops jararaca (jararaca).

A proteinase inhibitor was isolated from the plasma of Bothrops jararaca by three chromatographic steps: DEAE Sephacel, Phenyl Sepharose and Bio Gel P200. It inhibited caseinolytic and hemorrhagic activity of the whole venom of B. jararaca. Proteolytic activity of bothropasin and J protease, both metalloproteinases of the venom, were neutralized by the inhibitor. The J protease-inhibitor complex was isolated by gel filtration chromatography in HPLC and the electrophoresis pattern of this complex showed that the interaction between enzyme and inhibitor is not covalent.

Limited proteolysis of human and rabbit immunoglobulins by snake venoms produces Fab-like fragments.

Rabbit and, to a lesser extent, human immunoglobulins (IgGs) are cleaved by snake venoms. The snake venom proteases active on IgGs release fragments which behave on SDS-PAGE like Fab units similar to those released by the thiol-activated enzymes. However, in contrast to the cysteine proteases, the activity of snake venom proteases on IgGs is not only blocked by metal chelating agents but is also inhibited by thiol compounds. The snake enzymes active on IgG are metalloproteases.

Cleavage of immunoglobulins by moojeni protease A, from the venom of Bothrops moojeni.

Moojeni protease A, a proteolytic enzyme isolated from the venom of Bothrops moojeni hydrolyzes human and rabbit IgGs. The resulted fragments retained the combining though not the precipitating power or the property to fix complement. Similar to papain, moojeni protease A releases directly Fab fragments from IgG. In contrast to papain, however, the enzyme does not require the presence of thiol compounds either for activation or for reduction of the disulphide inter-heavy chain bridges. On the contrary, moojeni protease A is a metalloenzyme inhibited in the presence of thiol compounds.

Limited proteolysis of human and rabbit immunoglobulins by snake venoms produces Fab-like fragments

Rabbit and, to a lesser extent, human immunoglobulins (IgGs) are cleaved by snake venoms. The snake venom proteases active on IgGs release fragments which behave on SDS-PAGE like Fab units similar to those release by the thiol-activated enzymes. However, in contrast to the cysteine proteases, the activity of snake venom proteases on IgGs is not only blocked by metal chelating agents but is also inhibited by thiol compounds. The snake enzymes active on IgG are metalloproteases

Immunological comparison of hemorrhagic principles present in venoms of the Crotalinae and Viperinae subfamilies.

Antibodies were raised against hemorrhagic factors HF1, HF2 and HF3 isolated from the venom of Bothrops jararaca and NHFa,b from the venom of Bothrops neuwiedi. Crude venoms of different species of snakes were assayed with the rabbit antisera specific for the hemorrhagic factors. Results of immunodiffusion, neutralization of hemorrhagic activity and micro-complement fixation indicated that there is an immunological relationship between the venom hemorrhagic components of the Bothrops species and those of other species of the Crotalinae subfamily. The factors of Bothrops species seem to be structurally similar. The hemorrhagic proteins from the venoms of Lachesis, North American Crotalus, Asian Trimeresurus and Agkistrodon species show some resemblance to the Bothrops factors. The venom hemorrhagic principles from snakes of the Viperinae subfamily (Bitis and Vipera species) might have few epitopes similar to those of Bothrops species as the only relation shown was the partial neutralization by the immune sera.

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.

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.

Muscular lesions induced by a hemorrhagic factor from Bothrops neuwiedi snake venom.

The local tissue effects of crude Bothrops neuwiedi snake venom and of its hemorrhagic factor (NHF) were studied on mouse tibialis anterior muscle in vivo. After 6 h, 8 days and 6 weeks the muscles were examined in paraffin sections stained with hematoxylin and eosin. Both NHF and crude venom produced hemorrhage and myonecrosis, later followed by muscle fiber regeneration. Intramuscular arteries also suffered necrosis. The minimal dose of NHF necessary to produce detectable hemorrhage and myonecrosis was 50 ng, while the minimal venom dose needed to produce the same effect was 20 times higher. The results indicate that NHF is one of the major factors responsible for the local effects of B. neuwiedi venom.

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+.

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.

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.