Ribonuclease inhibitor from human placenta: interaction with derivatives of ribonuclease A.

Several specific modifications, both proteolytic and chemical, have been performed on RNase A. The ability of each of these RNase A derivatives to bind the human placental RNase inhibitor has been determined in competition binding assays. The interaction depends upon the native conformation of the enzyme. Loss of active site residues His-12 and His-119, along with auxiliary residues Lys-7, Phe-120, Asp-121, and Ser-123 in RNase S-protein, des-(121-124)-RNase, and des-(119-124)RNase, demonstrated that these residues are not essential for RNase-inhibitor binding. Carboxymethyl-Hisla-and carboxymethyl-His-119-RNase As interacted 3 times more strongly with the RNase inhibitor than did RNase A. Although ionic interactions between the basic RNase A (p1 = 9.4) and the acidic RNase inhibitor (p1 = 4.7) are involved, modification of the four arginine residues of RNase A by a&diketones does not alter the binding. Complete modification of the lysine residues of RNase A, with retention of positive charge through amidination, decreased but did not abolish the interaction. However, loss of positive charges through carbamylation of lysine residues decreased the interaction by 90% after only 3 lysine residues per molecule of enzyme were modified. Bound RNase inhibitor protected RNase A from inactivation by reagents which are known to inactivate the enzyme by reaction at Lys-41, a residue which appears to participate in the RNase-inhibitor binding and in the resultant inactivation of the enzyme.