Reactions of the amino groups in ribonuclease A: I. Kinetic studies

A kinetic analysis has been made of the reaction of the amino groups of ribonuclease A with trinitrobenzene-sulfonic acid. The number of reactive groups and the number of subsets were markedly dependent on the nature and concentrations of the buffer and the pH. Apparent values of pK_a, calculated from the variation of the velocity constants with pH, could, in general, be obtained only for pH values above 7.4. Below this pH the velocity constants were greater than the values calculated from the intrinsic constants. The values of pK_a were in the range of 7.9 – 9.0, which are somewhat smaller than those derived from titration data.The change of behavior of the amino groups with pH is confirmed by a study of the effects of ionic strength on the reactions.The velocity constants generally appear to decrease with increasing concentration of protein.It is shown that there is a close correlation between the pH region in which large changes occur of the reactivities of the amino groups of RNase and the kinetics of the enzyme reaction.