Mitsui N, Mitsui K, Hase J
Microbiol Immunol. 1980;24(7):575-84. doi: 10.1111/j.1348-0421.1980.tb02860.x.
Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/micrograms of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000 +/- 3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pIs, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly absorbed onto erythrocytes than theta-toxin of Cl. perfringens.
通过硫酸铵分级沉淀、丙酮沉淀和反复凝胶过滤，从破伤风梭菌菌株的培养液中纯化出破伤风溶血素。通过凝胶过滤分离出两种分子量不同的溶血素，其中较小的一种，即破伤风溶血素，得到进一步纯化。纯化使破伤风溶血素的比活性提高了1050倍，达到500 HU/微克蛋白质。纯化后的制剂在聚丙烯酰胺凝胶电泳上呈现出一条相对较宽的单一带，其活性与蛋白质浓度大致平行。然而，在十二烷基硫酸钠凝胶电泳上，它呈现出两条蛋白质含量几乎相等的带，分子量分别为53,000和48,000±3,000。此外，等电聚焦显示出活性的四个峰，其等电pH值按活性递减顺序分别为6.1、5.6、5.3和6.6。这些发现表明该制剂含有四种具有不同pI的溶血素，按分子大小可分为两组。该制剂完全不含破伤风神经毒素和蛋白酶。与产气荚膜梭菌的θ毒素相比，破伤风溶血素受胆固醇的抑制作用更强，在红细胞上的吸附速度更快。
