RESUMO
Experiments were performed to define the best isolation method for isolating Chrysaora fishing tentacle nematocyst organelles in order to minimize non-nematocyst contaminating proteins and proteases and stabilize crude nematocyst venom lethal activity. Techniques employed to disrupt the tentacles included autolysis, homogenization, or digestion using either trypsin or collagenase. Sephacryl-200 gel-filtration chromatography separated two lethal fractions. An immobilized serine protease inhibitor column, m-aminophenyl boronic acid acrylic beads, which reversibly bound one of the two lethal factors, was used in the second and third purification steps. By this means, a 105,000 mol wt. protein was purified, as judged by silver stained SDS-polyacrylamide gels. Lethal activity was inhibited by exposure to the serine protease inhibitor, L-1 chloro 3[4-tosylamido]-7-amino-2-heptanone-HCl, after purification. Although this lethal factor has some characteristics of a serine protease, it is not proteolytically active.
Assuntos
Ácidos Borônicos , Venenos de Cnidários/isolamento & purificação , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Aminoácidos/análise , Animais , Ácidos Borônicos/química , Cromatografia em Gel , Venenos de Cnidários/antagonistas & inibidores , Venenos de Cnidários/toxicidade , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Dose Letal Mediana , Camundongos , Microesferas , Urtiga-do-Mar da Costa Leste , Tosilina Clorometil Cetona/farmacologiaRESUMO
The enzyme, hyaluronidase, detected in crude venom was active over a pH range of 4-9 and was stable to at least a 72 hr storage at 4 degrees C. Preparative electrofocusing indicated a pI value for this enzyme between 9.5 and 10.2. Hyaluronidase partially purified by gel-filtration chromatography was evaluated as a spreading factor for dermonecrosis provoked by the nematocyst venom on depilated rats. The spread of dermonecrosis increased regardless of the presence or absence of hyaluronidase. Hemolytic activity of crude venom was assayed on erythrocyte suspensions from various sources. Pig and rat erythrocytes were the most sensitive to the hemolysin of the erythrocytes tested. The pH optima for the hemolysin was 8.3. Trypsinized rat erythrocytes were more susceptible to hemolysis induced by venom than non-trypsinized cells. The monovalent and divalent cations KCl, BaCl2, CaCl2, and MgCl2 were inhibitory to hemolytic activity induced by crude venom. The hemolysin partially purified by gel-filtration chromatography tested for stability after overnight storage at 4 degrees C and -70 degrees C indicated a 50% and 75% loss of activity, respectively, in comparison to the hemolytic activity recovered directly after gel-filtration chromatography.