Hydrogen ion equilibrai of staphylococcal enterotoxin B.

Potentiometric and spectrophotometric titrations of staphylococcal enterotoxin B have been carried out in 0.16 M KC1 at 25”. Potentiometric titration results indicate that the titration curves are essentially reversible between pH 1.50 and 11.0. All of the ionizable groups in enterotoxin B found from the titration curves are consistent with the amino acid composition which has been reported in the literature. The intrinsic pK values of individual ionizable groups and the electrostatic interaction factor, w, have been calculated. Spectrophotometric titration of enterotoxin B reveals that approximately 6 tyrosyl residues which have a pKi,t of 10.3 are titrated normally, while 8 tyrosyl groups are titrated between pH 11 and 12, and 7 tyrosyl residues are titrated between pH 12.0 and 13.5. Acetylation of enterotoxin B with acetylimidazole indicates that 5 to 6 “free” tyrosyl residues are present in enterotoxin B. Urea (8 M) had only a slight effect on the spectrophotometric titration and renders 10 tyrosyl groups available for acetylation. The tyrosyl groups are available for titration in 5 M guanidine-hydrochloride, however, only 14 tyrosyl residues are acetylated when lOOfold molar excess of acetylimidazole is used. All of the tyrosyl residues are available for acetylation with acetylimidazole in the presence of 5 M guanidine-hydrochloride when 300-fold molar excess of the reagent is used. Nitration of enterotoxin B with tetranitromethane revealed that 6 tyrosyl residues are available for the reaction. Essentially no loss of biological activity is observed when 6 free tyrosyl residues are chemically modified. However, antigen-antibody reaction was diminished when the abnormal tyrosyl residues were acetylated. Since conformational change has been observed after exposure of the abnormal tyrosine groups, the role of hydrophobic interactions for maintaining the rigid structure of enterotoxin B is discussed.