STAPHYLOCOCCAL ENTEROTOXIN C .I. PHENOLIC HYDROXYL IONIZATION

The ionization of the phenolic groups of enterotoxin C produced by Staphylococcus aureus strain 137 has been studied by spectrophotometric titration at 295 mμ in the range between pH 7.5 and 13.0. Of the 21 tyrosyl residues per mole of protein, 5 residues are deduced to be “free,” located on the surface of the molecule, freely exposed to the solvent and hence easily accessible to OH-. The ionization of these five “free” tyrosyl groups is reversible with PKapp= 10.02 and with no time dependence. The remaining 16 tyrosyl residues are postulated to be embedded in the interior (“buried”) and capable of ionizing only after unfolding of the protein molecule. The buried tyrosyl groups ionize at pH values higher than 11.0 and the ionization process is not reversible and is time dependent. Six of the buried ty rosyl residues ionize between pH 11.0 and 12.0 with a PKappof 11.5 and ten ionize above pH 12.0. Treatment of enterotoxin C with 5 m guanidine hydrochloride results in the normalization of all the 21 tyrosyl groups with a PKappof 9.9. The existence of two types of tyrosyl residues, free and buried, has also been demonstrated by studying the reaction of enterotoxin C with N-acetylimidazole, tetranitromethane, and tyrosinase. Five tyrosyl residues per mole of protein react with N-acetylimidazole at pH 7.5, five to six residues react with tetranitromethane at pH 8.0, and five groups are oxidized by tyrosinase at pH 6.5. © 1969, American Chemical Society. All rights reserved.