Isolierung und Charakterisierung von erythrogenen Toxinen I. Untersuchung des von Streptococcus pyogenes, Stamm NY-5 gebildeten Toxins A

Abstract

An erythrogenic toxin was isolated from culture supernatants of Streptococcus pyogenes, strain NY-5 (type 12) grown in yeast extract-pepton-dialysate medium. After concentration by evaporation, a crude material was obtained by precipitation with ethanol. This material was prepurified by ion exchange chromatography using a DEAE-Sepharose Cl-6B column. After precipitation of the active material with (NH4)2SO4 the redissolved precipitate was dialyzed against acetate buffer and rechromatographed on a CM-Sepharose Cl-6B column. The toxin was obtained by stepwise elution with 0.02 M acetate buffer, pH 5.0, and 0.05 M phosphate buffer, pH 6.5, followed by a last purification step on a Sephacryl S-200 column. The purified toxin behaved homogenously in SDS electrophoresis, the molecular weight being about 28 000. Amino acid analysis showed only one cysteine residue per molecule. The molecule was insensitive to 2-mercaptoethanol and alkylation. Isoelectric focusing yielded two narrow bands having an isoelectric point of 5.2. The toxin showed a serological reaction of identity with an antiserum against streptococcal pyrogenic exotoxin A (SPE-A) but was shown to have a different molecular weight. The purified material (erythrogenic toxin A) was pyrogenic and toxic for rabbits as well as mitogenic for human lymphocytes. A positive skin reaction in guinea pigs could be obtained by as little as 5 X 10(-8) mg.