Long-Rowe K O, Burnett J W
Department of Dermatology, University of Maryland, Baltimore 21201.
Toxicon. 1994 Apr;32(4):467-78. doi: 10.1016/0041-0101(94)90299-2.
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.
进行实验以确定用于分离金色海蜇触手刺丝囊细胞器的最佳分离方法，以便将非刺丝囊污染蛋白和蛋白酶降至最低，并稳定粗刺丝囊毒液的致死活性。用于破坏触手的技术包括自溶、匀浆或使用胰蛋白酶或胶原酶进行消化。Sephacryl-200凝胶过滤色谱法分离出两个致死组分。在第二和第三步纯化中使用了固定化丝氨酸蛋白酶抑制剂柱，即间氨基苯基硼酸丙烯酸珠，其可逆地结合两个致死因子之一。通过这种方法，经银染SDS-聚丙烯酰胺凝胶判断，一种105,000分子量的蛋白质被纯化。纯化后，暴露于丝氨酸蛋白酶抑制剂L-1氯3[4-甲苯磺酰胺基]-7-氨基-2-庚酮盐酸盐会抑制致死活性。尽管这种致死因子具有一些丝氨酸蛋白酶的特征，但它没有蛋白水解活性。
