The application of QAE-Sephadex for the purification of two staphylococcal enterotoxins. I. Purification of enterotoxin C2.
A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D004768:Enterotoxins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008722:Methods; D008970:Molecular Weight; D009994:Osmolar Concentration; D011817:Rabbits / Q000276:immunology; D013210:Staphylococcus / Q000032:analysis*; D014461:Ultracentrifugation
D000596:Amino Acids; D004768:Enterotoxins