Antonia P. Reichl

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.

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+.

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.

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 a myotoxin from the Duvernoy's gland secretion of the xenodontine colubrid Philodryas olfersii (green snake): Effects on striated muscle and the neuromuscular junction

A myotoxin has been isolated from the Duvernoys gland (DG) secretion of the xenodontine colubrid Philodrvas olfersii (green snake) by gel filtration on Sephadex G-100 SF. Under non-reducing and reducing conditions in SDS-PAGE, the myotoxin migrates as a single band with a mol. wt. of 20000. The toxin has 182 amino acid residues (approximately 20% acidic), a pI of 4.8 and a blocked N-terminal. In the chick biventer cervicis preparation, P. olfersii myotoxin partially blocks potassium-evoked contractures without affecting either the twitch-tension resulting from indirect stimulation or the contractures evoked by acetylcholine. Both the DG secretion and the myotoxin increase the serum creatine kinase (CK) levels of mice and stimulate the release of CK from the biventer cervicis preparation in a dose- and time-dependent manner. The varying degrees of muscle cell lysis and extensive widening of the intercellular spaces caused by the DG secretion are reproduced by the myotoxin, with the exception that in the latter the partial or total loss of transverse muscle striations is restricted to the muscle periphery. This myotoxin is the first such protein to be characterized from a DG secretion.

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).

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.

Biophysical properties and amino acid composition of Bothrops protease A, a proteolytic enzyme isolated from the venom of the snake Bothrops jararaca (jararaca)

Bothrops protease A, an arginine-ester hydrolase, is active on protamine, gelatin and insulin and was isolated from the venom of Bothrops jararaca in a homogeneous state, as judged by polyacrylamide gel electrophoresis and ultracentrifugal analyses. The enzyme has a molecular weight of 65,000 and a pI of 3.55. The enzyme is a glycoprotein whose amino acid content corresponds to 55% of the molecular weight.

Isolation, characterization and biological properties of two kinin-like peptides (peptide-S and peptide-R) from Scaptocosa raptoria venom

Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.