Beaudet R, McSween G, Bisaillon J G
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Laval, Canada.
Res Vet Sci. 1993 Jan;54(1):45-51. doi: 10.1016/0034-5288(93)90009-5.
An unstable haemolytic activity produced by a strain of serotype 1 of Actinobacillus pleuropneumoniae was isolated when 1 per cent bovine serum albumin (BSA) was added to RPMI 1640 medium. BSA acts as a carrier molecule and stabilises activity. This haemolysin (BSA-haemolysin) was precipitated with ammonium sulphate, dialysed and lyophilised. Of the species tested, bovine erythrocytes were the most susceptible to the BSA-haemolysin while mouse and rabbit erythrocytes were the least susceptible. The haemolytic activity was dependent on the incubation temperature, no activity being observed at or below 24 degrees C. The haemolytic activity was also partly stabilised by 100 micrograms ml-1 dithiothreitol (DTT). The DTT-haemolysin was purified to homogeneity by ultrafiltration and high pressure liquid chromatography on a Protein Pak DEAE-5PW column. The molecular weight was estimated at 16 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and at 23 kDa by molecular gel filtration from the elution position of the haemolytic activity. The DTT-haemolysin activity was completely destroyed by pronase treatment suggesting that this substance could be a polypeptide. The addition of BSA to DTT-haemolysin increased its activity and stability to lyophilisation. The addition of 10 mM calcium chloride in the titration assay increased the activity of DTT-haemolysin from 220 to 476 haemolytic units ml-1. The BSA-haemolysin activity was only slightly affected by the addition of calcium chloride.
当向RPMI 1640培养基中添加1%的牛血清白蛋白（BSA）时，分离出了胸膜肺炎放线杆菌血清型1菌株产生的一种不稳定溶血活性。BSA作为载体分子，可稳定该活性。这种溶血素（BSA - 溶血素）用硫酸铵沉淀、透析并冻干。在所测试的物种中，牛红细胞对BSA - 溶血素最敏感，而小鼠和兔红细胞最不敏感。溶血活性取决于孵育温度，在24℃或更低温度下未观察到活性。100微克/毫升二硫苏糖醇（DTT）也能部分稳定溶血活性。通过超滤和在Protein Pak DEAE - 5PW柱上进行高压液相色谱法将DTT - 溶血素纯化至同质。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计其分子量为16 kDa，通过根据溶血活性的洗脱位置进行分子凝胶过滤估计为23 kDa。经链霉蛋白酶处理后，DTT - 溶血素活性完全被破坏，表明该物质可能是一种多肽。向DTT - 溶血素中添加BSA可提高其活性以及冻干稳定性。在滴定试验中添加10 mM氯化钙可使DTT - 溶血素的活性从220溶血单位/毫升增加到476溶血单位/毫升。添加氯化钙对BSA - 溶血素活性的影响很小。
