Reactions of the amino groups of RNAse A: IV. The effects of protein concentration

A study has been made of the effect of ribonuclease (RNAse) concentration on the properties of the amino groups. The biphasic dependence of pK on pH which has been established (Goldfarb and Martin, Bioorg. Chem.5, 137 (1976)). for 5 μM solution of RNAse also have been shown to occur for 50 μM solutions. In the lower pH range (7.5–8.5) the values of pK obtained with 50 μM solutions were similar to those obtained with 5 μM solutions (pK = 7.5) but the intrinsic constants were smaller. In the higher pH range (8.5–10) the pKs in the more concentrated solutions were larger than those found at the smaller concentration and the intrinsic constants were generally smaller. A quantitative study of the concentration vs k_i relation at pH 7.5 indicated a sigmoid relationship for all of the subsets with a constant maximum value equal to, and less than that at 5 μM RNAse and a constant minimum value above that at 20 μM. Parallel studies with oxidized RNAse gave parallel, although not identical, results from which it is proposed that the concentration effect does not arise totally from the three-dimensional structure of native RNAse.