Date of this Version
The visible and ultraviolet circular dichroic (CD) spectra resulting from the interaction of ribonuclease with successive Cu(I1) ions have been recorded under a variety of conditions. At pH 7 in the presence of 0.16 M KC1 a broad, negative band was found in the visible region. This band increased in intensity and changed in shape as successive coppers were added. The circular dichroic spectra could be analyzed in terms of two kinds of binding sites: a single strong site with CD minimum at about 710 nm, and four weaker sites with CD minimum at about 600 nm. The binding constants observed are close to those obtained by more conventional means. Carboxymethylation of one histidine results in loss of one of the weaker sites. In 0.01 M salt, only the 600-nm band is seen. Binding at pH 9.6 differed in that saturation did not occur until about 33 sites had been filled. The presence of tetra coordination at this pH was indicated by the shift of the primary d-d transition down to 530 nm. Additional structure in the visible and near ultraviolet CD was now present in the form of a negative band at 355 nm and, for the first two Cu(II)‘s added, a positive one at 480 nm. Strong positive bands were observed at 251 and 305 nm for all pH values ≥7. These are tentatively ascribed to charge transfer complexes between Cu(I1) and the peptide backbone. The relationship of the Cu(II)-ribonuclease CD spectra to those of natural, copper-containing metalloproteins, both “blue” and “non-blue”, is discussed, with special emphasis on the oxyhemocyanins.