Spectrophotometric titration of phenolic groups of pepsin.
The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.
D000818:Animals; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A; D010636:Phenols; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014443:Tyrosine; D014783:Viscosity
D006146:Guanidines; D010636:Phenols; D014443:Tyrosine; D010434:Pepsin A