Purity of Staphylococcal  -haemolysin obtained by three different procedures

Abstract

To S T U D Y the biological, physical, and chemical characteristics of staphylococcal 6-haemolysin, and also to determine its role in host-parasite interactions we needed to obtain a purified preparaton of S-haemolysin. At present, three methods (Wiseman and Caird, 1968 ; Kreger, Zabovetzky and Bernheimer, 1971 ;Kantor, Temples and Shaw, 1972) have been reported for purifying 6-haemolysin. In this investigation we tried to compare these methods and to test the purity of the 8-haemolysin preparations so obtained. Our findings indicate that when biological tests were used for establishing the purity of the final products none of the methods tested yielded a pure preparation of S-haemol y sin.MATERIALS AND METHODS Strain. The 146P strain of Staphylococcus aureus previously described (Murphy and Haque, 1971) was used.Crude 8-toxin. This was obtained by growing the culture on dialysis membranes as previously described (Murphy and Haque, 1971).Identification of haemolysins. The various haemolysins were identified by the electrophoretic localisation technique (Haque, 1967). 6-Haemolysin was defined as the haemolytic moiety that upon electrophoresis in a 1 % gel of Ion Agar no. 2 (Oxoid) in barbital buffer (PH 8.4) at 5 mA per cm for 13 h, remained at the origin and lysed human, rabbit and horse erythrocytes. a-Haemolysin under these same conditions migrated 18-20 mm toward the cathode and lysed rabbit, sheep and horse erythrocytes, and in concentrated preparations it lysed human erythrocytes. 6-Haemolysin migrated approximately 36 mm toward the cathode and lysed sheep erythrocytes only.Measurement of 8-haemolysin. The tube titration procedure as previously described (Murphy and Haque, 1971) was used. The haemolytic titre was the highest dilution of a haemolytic sample that gave 50 % Iysis of a 1 % suspension of washed human erythrocytes and the titre represented the number of haemolytic units (HU) in the sample. Specific activity of

Partitioning of staphylococcal haemolysin

“…In addition, the chromatographic procedures employed frequently yielded multiple peaks of 6 haemolysin, while crude preparations yielded only one peak of S haemolysin when tested electrophoretically and by molecular sieving. These findings, as well as those previously reported (Fackrell and Wiseman, 1974;Lee et al, 1976) clearly indicate the need to renew efforts to develop methods for the purification of 6 haemolysin.…”

“…The production, identification, and characterisation of the crude toxin and the analysis for biological activities of the preparations obtained by the various fractionation procedures were carried out as previously described (Haque, 1967;Murphy and Haque, 1967Murphy, 1972;Lee et al, 1976;Madler et al, 1976).…”