Studies on extracellular proteins from Staphylococcus aureus. IV. Separation of α-toxin by isoelectric focusing

Abstract

Three strains of Staphylococcus aureus (V8, M18, and Wood 46) were grown in batch and continuous culture in ‘CCY medium’. Hundred-fold differences in toxin production were found for strain V8 and Wood 46 when the bacteria were grown to a density of 6–8 g/l (dry weight). Dialyzed culture supernatants and crude concentrated toxin preparations were subjected to isoelectric focusing in pH gradients. The α-toxin turned out to be separable into three or four components with different isoelectric points (p I's). The main component, α Ia, was found to contain roughly 80% of the total haemolytic activity and had a p I of 8.5. The carrier ampholytes and the sucrose had a stabilizing effect on the toxins. Yields of 70–140% were calculated. The degree of purification and the specific activities are estimated. Electrofocusing experiments were performed in 6 M urea and gave analogous results which was also the case for ‘heat-purified α-toxin’. After Sephadex G-75 chromatography one single peak of α-toxic activity was found. Finally, isoelectric focusing of single fractions of α Ia IIIa showed an interconversion phenomenon. These studies suggest that the multiple forms of α-toxin differ in conformation.