Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest.

A comparative study of venoms from juvenile, sub-adult and adult wild Bothrops atrox specimens captured in Manaus region (Brazil) was performed. All venoms tested had acidic pH (5.5) and the human plasma coagulant activity was higher in venoms from juvenile and sub-adult specimens than in adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the most intense bands in adult venoms corresponded to polypeptides of 23 and 50kDa. The 23kDa protein was not detected in juvenile venoms. The 23 and 50kDa proteins were purified by two steps of reversed phase-HPLC followed by size exclusion HPLC. Partial amino acid sequence of the 23kDa protein showed homology to metalloproteinases from other snake venoms. Electrospray ionization mass spectrometric analysis (ESI-MS) showed that the 23kDa band contained at least three isoforms of 23030, 23300 and 23645Da. The 50kDa polypeptide was N-terminally blocked for Edman degradation and presented molecular masses ranging from 46.8 to 49.4kDa by ESI-MS. Both proteins were detected by anti-mutalysin II antibodies in immunoblotting assay indicating that they belong to the metalloproteinase family. Immunoblotting analysis also showed that the 23kDa band increased in intensity from juvenile to adult specimens.SDS-PAGE analysis of juvenile and adult venoms following autoproteolysis in pH 7.4 suggested that endogenous venom metalloproteinases can digest the 50kDa metalloproteinase, originating a new protein band of 27kDa. It was also demonstrated in juvenile venoms that the 23kDa band was not the result of proteolytic processing of the 50kDa metalloproteinase.

Cytolytic proteins and peptides

É apresentada uma revisäo sobre proteínas e peptídios citolíticos encontrados em bactérias, fungos, invertebrados marinhos, artrópodes, répteis, citolítocs encontrados em bactérias, fungos, invertebrados marinhos, artrópodes, répteis,, anfíbios, humanos e plantas. Ela pöe em evidência um novo grupo de proteínas e peptídos de plantas

Enterolobin, a hemolytic protein from Enterolobium contortisiliquum seeds (Leguminosae - Mimosoideae): purification and characterization

Hemolytic and phospholipase D activities were found in the saline extract of Enterolobium contortisiliquum seeds. The hemolytic activity is due to a protein which was named enterolobin. This protein was highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of saturation, batch separation by adsorption on DEASE - cellulose and gel filtration chromatography on Sephadex G-100 or G-150. In the batch separation the fraction showing hemolytic activity was not adsorbed by the resin while the fraction with phospholipase activity was. In this manner it was shown that those two activities were due to different proteins. Mouse erythrocytes were less susceptible to hemolysis by enterolobin than human and rabbit erythrocytes. The hemolytic activity was rapidly lost at or above 55§C and in extreme acid (1.6) and basic (10.8) pHs. The following characteristics of purified enterolobin were determined: molecular weights of 55.000 D (by SDS-PAGE), 59.800 D (by gel filtration) and 51.300 D (by HPLC); pI=7,0; Gln as the N-terminal amino acid residue; high levels of Asp(Asx), Glu(Glx), Ser and Thr residues and low levels of Cys and Met residues. Similarities were noticed between enterolobin and crotin, a hemolytic protein of Croton tiglium seeds

The complete amino acids sequence of the Vigna unguiculata (L.) Walp. seed trypsin and chymotrypsin inhibitor

Foi determinado em nosso Laboratório a seqüência completa de aminoácidos de um inhbidor de tripsina e quimotripsina, "double-headed", denominado abreviadamente BTCI, purificado de feijäo caupi Vigna unguiculata (L.) Walp.cv."Seridó". Os peptídeos trípticos e quimiotrípticos foram seqüenciados pelos métodos manuais de Edman-Gray, de Edman-Chang (N-terminais) e de carboxypeptidases (C-terminais). É a seguinte a seqüência de aminoácidos do BTCI: Ser Gly-His-Glx-Asx-Ser-Thr-Asx-Glx-Ala-Ser-Glx-Ser-Ser-Lys-Pro-Cys-Cys-Arg-Glx-Cys-Ala-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Glx-Cys-Arg-Cys-Ser-Asx-Val-Arg-Leu-Asn-Ser Cys-His-Ser-Ala-Cys-Lys-Ser-Cys-Thr=Phe-Ser-Ile-Pro-Ala-Glx-Xys-Phe-Cys-Gly=Asx-Ile-Asx-Asx-Phe-Cys=Tyr-Lys-Pro-Cys-Lys-Ser-Ser-His-Ser-Asx-Asx-Asx-Asx0Trp-Asn. BTCI apresenta alto grau de homologia como vários outros inibidores da família Bowman-Birk