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The interaction of ribonuclease with metal ions. IV. Role of the alpha-amino group in cupric ion binding.

Abstract A derivative of pancreatic ribonuclease, in which the terminal α-NH2 has been converted to a hydroxyl group, has been prepared by treatment with nitrous acid. Comparative potentiometric titrations of this derivative and of unmodified RNase indicate the assignment at 25° of a pK of 7.3 to the α-NH2 of RNase. The Cu(II)-binding properties of RNase lacking the terminal α-NH2 have been compared with those of unmodified RNase by spectrophotometric and gel filtration binding studies. Analysis of gel filtration data obtained at pH 7 in 0.4 m acetate is most consistent with a weak cooperativity among the binding sites of both RNase and its deaminated derivative. Differences between the two proteins in these studies indicate that loss of the α-NH2 from RNase leads to loss of the strongest of its five Cu(II)-binding sites at pH 7. Spectrophotometric studies in the absence of acetate at pH 7 show that the 640 mµ absorption band of the 1:1 Cu(II)-RNase complex is diminished in intensity by 35% in the corresponding complex of deaminated RNase. Between pH 7 and pH 9, the net spectral contribution of the site lost by deamination shifts from 640 mµ to 540 mµ. The data support the thesis that the single strongest Cu(II)-binding site in RNase under these conditions contains the α-NH2 and its adjacent peptide bond nitrogen atoms. At pH 5.5, gel filtration and spectrophotometric differences between the deaminated derivative and RNase are of smaller magnitude than at pH 7 and indicate that the α-NH2 is one of the weaker Cu(II)-binding sites at this pH. Formation of the ternary complex involving Cu(II), RNase, and 3'CMP has been shown to be independent of the presence of the α-NH2 terminus.