A simplified method was devised for the purification of staphylococcal enterotoxin A on a large scale. In it, a supernatant fluid from the 48-hour culture of
Staphylococcus aureus 13 N-2909 in 4% NZ-amine was subjected directly to chromatography on Amberlite CG-50 at first, then to CM-cellulose column chromatography, and finally to Sephadex G-75 gel filtration. The purified toxin preparation obtained was studied for physicochemical properties. purified toxin preparation obtained was studied for physicochemical properties.
As a result, 380 mg of enterotoxin A, with a purity greater than 99%, was obtained from 80 liters of culture filtrate. The overall recovery of the toxin was calculated to be about 36%. The purified preparation gave a maximum absorption at 277 nm and a minimum absorption at 250 nm. It was proved both immunologically and physicochemically that the toxin was homogeneous protein. No nucleic acid, carbohydrate, or lipid was detected in the toxin. The sedimentation coefficient (
S20w) of the purified toxin was 2.71 S. Its molecular weight was estimated to be 26, 000 by gel filtration on Sephadex G-75, 27, 000 by SDSpolyacrylamide gel electrophoresis, and 30, 000 by sedimentation equilibrium.
The presence of two major components was demonstrated by the isoelectric focusing technique. The isoelectric point of the principal component was determined to be pH 7.0 at 20 C. The toxin showed the 50% loss of its serological reactivity when heated at 60 C for 3 hours or at 80 C for 5 hours. It was completely inactivated when heated at 100 C for 2 hours. Amino acid analysis revealed that the toxin consisted of 214 amino acid residues.
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