Hydrogen Ion Titration of Ionizable Side‐chains in Native and Denatured GLYCI.NIN

Electrometric and spectrophotometric titration curves of glycinin were obtained at 0.4 ionic strength and in the presence of 6M guanidine hydrochloride and 6M urea. The electrometric hydrogen ion titration curves were irreversible in the pH range 6.5 to 12.0 in 0.4M KCl, but exhibited reversibility in the pH 2.0 to pH 6.5 range. The forward and backward electrometric titration curves were entirely reversible in 6M guanidine hydrochloride. The spectrophotometric titration curves of tyrosine groups were also irreversible in 0.4M KCl, but reversible in 6M urea. Alkali‐induced denaturation produced a different spectrophotometric titration curve than the curves obtained with glycinin in 0.4M KCl and 6M urea. The electrostatic factor w was lower in the pH 2.50 to 3.75 range than in the pH 3.75 to 6.50 range. This was interpreted to be due to swelling and dissociation into subunits of the glycinin molecule subjected to acid denaturation. The empirical value of w = 0.045 derived from the titration of the carboxyl groups in the pH 3.75 to 6.50 range coincided with the theoretically calculated value of 0.040 for a swollen sphere. Group counting and pK values of carboxyl, imidazole, e‐amino, and phenoxy groups under normal and denaturing conditions are also reported.