The Homologous and Hybrid Dimers of Ribonuclease A and Its Carboxymethylhistidine Derivatives

Abstract

Abstract The formation of easily dissociable dimers by lyophilization from 50% acetic acid, first demonstrated with ribonuclease A, has now been shown to occur with similar yields with either 3-carboxymethylhistidine-12 ribonuclease (3-CMHis-12-RNase) or 1-carboxymethylhistidine-119 ribonuclease (1-CMHis-119-RNase). Lyophilization of a mixture of monomers, such as ribonuclease A and 1-CMHis-119-RNase or 3-CMHis-12-RNase and 1-CMHis-119-RNase, yields a dimer fraction which, as expected from earlier work, is a mixture of homologous and hybrid dimers. Each type of dimer, whether homologous or hybrid, is resolved by ion exchange chromatography into two isomers, Forms I and II, analogous to those reported for the dimer of ribonuclease A. The hybrid dimers of ribonuclease A and 1-CMHis-119-RNase, in both Forms I and II, were prepared by two routes: (a) by lyophilization of the mixed monomers and (b) by alkylation of each of the dimers of ribonuclease A. The catalytic and chromatographic properties of the dimers did not depend on the route of formation. Alkylation did not result in isomerization between Forms I and II. A slight difference in conformation involving areas of the molecule remote from the active site remains the most likely explanation for the existence of these two isomers. In addition to gel filtration on Sephadex G-75 and chromatography on sulfoethyl Sephadex in phosphate buffers, rapid chromatography on Amberlite IRC-50 with tris(hydroxymethyl)aminomethane buffer has been used in the resolution of dimers. A comparison of the stabilities of some dimers shows that, at pH 7.45, the presence of a 3-CMHis-12 residue tends to destabilize the molecule, whereas the presence of a 1-CMHis-119-119 residue exerts a stabilizing effect. Each hybrid dimer of ribonuclease A and 1-CMHis-119-RNase and of 3-CMHis-12-RNase and 1-CMHis-119-RNase has 50 ± 5% of the specific activity of ribonuclease monomer toward 2',3'-cyclic cytidylate. This supports the postulate that each of these dimers has one reconstructed active site with essentially full activity.