Albesa I, Barberis L I, Pájaro M C
Departamento de Microbiología de Inmunología, Facultad de Ciences Exactas, Universidad Nacional de Río Cuarto, Provincia de Córdoba, Argentina.
Rev Argent Microbiol. 1985;17(1):33-9.
The hemolytic activity of sixty K. pneumoniae strains was investigated in tryptic soy agar with rabbit, dog, sheep, human, chicken and mouse blood. All of them were lytic only for rabbit red cells. In liquid medium it was necessary a 2 mercaptoethanol treatment to detect a good degree of hemolysis. Cultures in tryptic soy broth gave 100% hemolysis in assays with rabbit erythrocytes and only when hemolysin was concentrated by purification was it active on dog and sheep but in a lesser degree (7.5% hemolysis). Supernatants of cultures were precipitated at different saline concentrations; the fraction obtained with 30-50% (NH4)2SO4 had hemolytic activity after dialysis and 2 mercaptoethanol treatment. Then this fraction was eluted in a Sephadex G-100 column, but electrophoresis in polyacrylamide gel showed that the hemolytic molecules obtained by gel filtration were contaminated with protein structures which had different electrophoretic migration. Ion-exchange chromatography showed best purification index and the recovery of activity was over 100%, it was possible to explain this good recovery once an inhibitor was detected. Two rabbit red cell lysins were purified, both shared several properties: SH-activation, pH optimum, thermolability, selectivity for rabbit red cells, mechanism of action, inhibition by cholesterol and divalent cations.
在胰蛋白胨大豆琼脂培养基中，用兔、犬、羊、人、鸡和小鼠的血液对60株肺炎克雷伯菌的溶血活性进行了研究。所有菌株仅对兔红细胞具有溶血作用。在液体培养基中，需要用2-巯基乙醇处理才能检测到良好程度的溶血。胰蛋白胨大豆肉汤培养物在兔红细胞试验中溶血率达100%，只有当溶血素通过纯化浓缩后，才对犬和羊红细胞有活性，但程度较低（溶血率7.5%）。培养物的上清液在不同盐浓度下沉淀；用30 - 50%硫酸铵获得的部分经透析和2-巯基乙醇处理后具有溶血活性。然后将该部分在Sephadex G - 100柱上洗脱，但聚丙烯酰胺凝胶电泳显示，通过凝胶过滤获得的溶血分子被具有不同电泳迁移率的蛋白质结构污染。离子交换色谱显示出最佳的纯化指数，活性回收率超过100%，一旦检测到一种抑制剂，就可以解释这种良好的回收率。纯化了两种兔红细胞溶血素，它们具有几个共同特性：SH激活、最适pH、热稳定性、对兔红细胞的选择性、作用机制、受胆固醇和二价阳离子抑制。
