Purification of cereolysin and the electrophoretic separation of the active (reduced) and inactive (oxidized) forms of the purified toxin.
Cereolysin was purified to apparent homogeneity by using ammonium sulfate fractionation, hydrophobic chromatography with AH-Sepharose, isoelectric focusing, and gel filtration. The active form of the toxin had an isoelectric point of 6.6, and the molecular weight of the protein was about 55,500 as judged by sodium dodecyl sulfate-gel electrophoresis, gel filtration, and gel electrophoresis using various concentrations of acrylamide. Cereolysin contained two half-cystine residues and was dependent on reducing agents, such as dithiothreitol, for maximal hemolytic activity and charge homogeneity. By using discontinuous acrylamide electrophoresis, two forms of the toxin could be observed: oxidized and reduced. If the toxin was purified in the absence of dithiothreitol, partial spontaneous oxidation resulted in the formation of an oxidized form of the toxin. Relative to the reduced form, the oxidized form moved slightly closer to the anode in gel electrophoresis at pH 9.0. If the toxin was purified in the presence of 5 mM dithiothreitol or if the spontaneously oxidized toxin was preincubated with dithiothreitol, only the reduced form of the protein was observed. When the logarithims of their relative mobilities were plotted against the concentration of acrylamide in the gels, the slopes for the reduced and oxidized forms were identical. This indicates that the two forms are identical in size and are separable because of different charges. The reduced protein could be inhibited by N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-hydroxymercuribenzoate. Inhibition by the latter two sulfhydryl reagents could be completely reversed by dithiothreitol. The reversibly oxidized form of the toxin did not appear to be inhibited by N-ethylmaleimide and apparently was either unable to bind to or had a decreased affinity for the erythrocyte membrane.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001409:Bacillus cereus / Q000032:analysis*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis / Q000187:drug effects; D007525:Isoelectric Focusing; D008970:Molecular Weight; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014118:Toxins, Biological / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology
D000596:Amino Acids; D013439:Sulfhydryl Reagents; D014118:Toxins, Biological; D000645:Ammonium Sulfate; D004229:Dithiothreitol