Gow J A, Robinson J
J Bacteriol. 1969 Mar;97(3):1026-32. doi: 10.1128/jb.97.3.1026-1032.1969.
Purification of beta-hemolysin was achieved by ammonium sulfate precipitation, Sephadex G-100 gel filtration, carboxymethyl cellulose column chromatography, and density gradient electrophoresis. Active fractions eluted from carboxymethyl cellulose contained at least one nonhemolytic protein, and omission of this step was not detrimental to the purification process. Density gradient electrophoresis yielded approximately 1.6 mg of highly active purified beta-hemolysin per liter of culture supernatant liquid. Purified beta-hemolysin gave a single line on gel double diffusion and immunoelectrophoresis. A single symmetrical peak formed in the analytical ultracentrifuge, and the sedimentation coefficient was calculated to be 1.7S. The purified beta-hemolysin was stable at 4 C and could be lyophilized. Magnesium cations were required for full expression of beta-hemolytic activity. beta-Hemolysin was lethal for rabbits when injected intravenously in amounts between 40 and 160 mug. Crude beta-hemolysin was more stable than purified beta-hemolysin when heated at 60 C for 30 min. Purified beta-hemolysin lost almost all of its activity on subsequent heating at 100 C for 10 min.
通过硫酸铵沉淀、葡聚糖G - 100凝胶过滤、羧甲基纤维素柱色谱和密度梯度电泳实现了β - 溶血素的纯化。从羧甲基纤维素洗脱的活性组分含有至少一种非溶血蛋白，省略这一步骤对纯化过程无害。密度梯度电泳每升培养上清液可产生约1.6毫克高活性的纯化β - 溶血素。纯化的β - 溶血素在凝胶双向扩散和免疫电泳中呈现单一沉淀线。在分析超速离心机中形成单一对称峰，沉降系数计算为1.7S。纯化的β - 溶血素在4℃下稳定且可冻干。β - 溶血素的完全溶血活性需要镁离子。当以40至160微克的量静脉注射时，β - 溶血素对兔子具有致死性。粗制β - 溶血素在60℃加热30分钟时比纯化的β - 溶血素更稳定。纯化的β - 溶血素在随后100℃加热10分钟后几乎丧失了所有活性。
