Purification of a lethal factor and characteristics of hyaluronidase and hemolytic activity in the nematocyst venom of the sea nettle, Chrysaora quinquecirrha

Abstract

There are at least two lethal factors to mice in crude venom preparations of the nematocysts isolated from the fishing tentacles of the sea nettle, Chrysaora quinquecirrha. Crude venom obtained from nematocysts isolated by tentacle homogenation was used in a purification scheme derived from experiments of screening various protease inhibitors to remove endogenous proteases. An immobilized protease inhibitor column, m-aminophenyl boronic acid acrylic beads, was found to reversibly bind one of the lethal factors. A 105 Kd protein was enhanced 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 (TLCK) after the three stages of purification. Western transfers of the lethal fraction to nitrocellulose after treatment to detect the presence of glycoconjugates indicated that this protein was not glycosylated. The enzyme, hyaluronidase, detected in crude venom was active over a pH range between 4-9 and was stable to at least a 72 h storage at 4{dollar}\sp\circ{dollar}C. Preparative electrofocusing indicated a pI value for this enzyme between 9.5-10.2. Hyaluronidase partially purified by gel-filtration chromatography was evaluated as a spreading factor for dermonecrosis induced 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, BaCl{dollar}\sb2{dollar}, CaCl{dollar}\sb2{dollar}, and MgCl{dollar}\sb2{dollar} 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{dollar}\sp\circ{dollar}C and {dollar}-{dollar}70{dollar}\sp\circ{dollar}C indicated a 50% and 75% loss of activity, respectively, in comparison to the hemolytic activity recovered directly after gel-filtration chromatography. (Abstract shortened with permission of author.)