Optical Activity of Cystine-containing Proteins

Circular dichroic (CD) spectra are reported for pancreatic ribonuclease A which has been reoxidized under various conditions, and for partially reduced forms of RNase A. Reoxidation of reduced RNase A in the absence of -mercaptoethanol leads to a product averaging 83% of the enzymatic activity of native RNase but differing in CD spectral bands, most prominently in the vicinity of 240 mii where native RNase exhibits a positive band and the reoxidized material does not. If the reoxidation is carried out in the presence of 03-mercaptoethanol, resultant enzymatic activities are about the same, but the anomalous CD spectrum near 240 mu is not observed. RNase which has been reoxidized in the absence of -mercaptoethanol can be fractionated on carboxymethyl Sephadex to yield two components, designated CMS-I and CMS-II. CMS-I is enzymatically inactive. CMS-I is fully active toward RNA. Its CD spectrum is identical with that of native RNase everywhere except near 240 my. With CMS-II, the positive band is present, but its intensity is only about half that of native RNase. The differences are amplified by cooling to below 5 . Calculated values of molecular ellipticity at 240 my for mixtures of CMS-I and CMS-II are similar to those observed with unfractionated reduced-reoxidized RNase; calculated values for mixtures of CMS-I and native RNase do not agree with the observed spectra. RNase, which, on the average, has one disulfide bond reduced, can be air-reoxidized in the absence of -mercaptoethanol to a material whose CD spectrum is identical with that of RNase A. More extensive reduction, to an average of 4 sulfhydryl residues per molecule, yields on reoxidation a protein whose CD spectrum lacks the positive band at 240 mi.