The Interaction of Ribonuclease with Metal Ions

• Published in: The Journal of biological chemistry Vol. 245; no. 7; pp. 1527 - 1536
• Main Authors: Breslow, Esther, Girotti, Albert W.
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 10.04.1970
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)77127-0

The effect of 2'-cytidylic acid and 3'-cytidylic acid upon the binding of cupric ions by RNase has been studied by gel filtration, together with the effect of cupric ions upon binding of 2'- and 3'-cytidylic acids. The results confirm that binding of 2'-CMP by RNase weakens its affinity for cupric ions; reciprocally, binding of cupric ion diminishes the affinity of RNase for 2'-CMP. The negative interactions between cupric ion and 2'-CMP upon binding to RNase have been shown to lead to distortion of the gel filtration ligand trough. At pH 5.5 these interactions are tentatively interpreted in terms of competition between 2'-CMP and Cu(II) for the strongest Cu(II)-binding site on RNase together with an acetate-dependent increase in the Cu(II) affinity of one of the weaker RNase sites in the presence of bound 2'-CMP. Binding of 3'-CMP to RNase increases the affinity of RNase for 2 cupric ions; similarly binding of cupric ions to RNase increases its affinity for 3'-CMP. Analysis of the binding pattern of Cu(II) to the 3'-CMP-RNase complex indicates co-operative interactions between two Cu(II)-binding sites on the 3'-CMP-RNase complex; at least one of these sites differs from any on free RNase and could involve the phosphate group of 3'-CMP. In further contrast to free RNase, the Cu(II)-binding sites at pH 5.5 on the 3'-CMP-RNase complex have been shown to have a diminished affinity for the cupric ion-monoacetate complex relative to free cupric ion. This suggests that, on the average, Cu(II) is coordinated with more ligands on the 3'-CMP-RNase complex than on free RNase or, alternatively, that binding of Cu(II) to the complex occurs in a more sterically limited environment than on free RNase.