Production and purification of a low molecular weight haemolysin produced by Actinobacillus pleuropneumoniae serotype 1

Abstract

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 haemolytin ( bsa-haemolytin) was precipitated with ammonium sulphate, dialysed and lyophilised. Of the species tested, bovine erythrocytes were the most susceptible to the bsa-haemolytin 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°C. The haemolytic activity was also partly stabilised by 100 μg ml −1 dithiothreitol ( dtt). The dtt-haemolysin was purified to homogeneity by ultra-filtration 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-haemolytin from 220 to 476 haemolytic units ml −1. The bsa-haemolytin activity was only slightly affected by the addition of calcium chloride.