Barberis L I, Eraso A J, Pájaro M C, Albesa I
Can J Microbiol. 1986 Nov;32(11):884-8. doi: 10.1139/m86-161.
Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis. The molecular weights, estimated by gel filtration, were 8400 and 19,000; by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, the values were calculated as 15,500 and 27,000. The electrophoretic bands were best detected by the periodic acid--Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19,000 hemolysins. However, trypsin treatment cleaved the 19,000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19,000 hemolysin and the smaller hemolysin was absent.
通过盐析、凝胶过滤、离子交换色谱和聚丙烯酰胺凝胶电泳等方法，从生长培养基中纯化出两种硫醇激活的肺炎克雷伯菌溶血素。通过色谱法和电泳法测定，溶血素峰与蛋白质和糖蛋白峰一致。通过凝胶过滤估计的分子量分别为8400和19,000；通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳计算，其值分别为15,500和27,000。电泳带通过高碘酸-希夫法最易检测到。二硫键的还原并没有使原本较大的分子裂解成8400和19,000的溶血素。然而，胰蛋白酶处理将19,000的溶血素裂解成一个活性部分，其电泳迁移与8400的溶血素相似。使用胃蛋白酶抑制剂和抗蛋白酶研究了一种天然存在的蛋白水解活性。当向系统中加入胰蛋白酶抑制剂时，仅在19,000的溶血素中检测到溶血活性，而较小的溶血素不存在。
