PRODUCTION AND PURIFICATION OF A LOW-MOLECULAR-WEIGHT HEMOLYSIN PRODUCED BY ACTINOBACILLUS-PLEUROPNEUMONIAE SEROTYPE-1

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 mug 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.