Fractionation of Ribonuclease A Photosensitized with 4-Thiouridylic Acid

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Abstract
Bovine pancreatic ribonuclease A [EC 2.7.7.16] was inactivated by irradiation with near-ultraviolet light in the presence of 4-thiouridylic acid (F. Sawada, J. Biochem., 65, 767 (1969)). The irradiated enzyme was fractionated into two classes: partially inactivated monomers and inactivated aggregates. The monomers were composed of at least 6 components. It seemed that cystinyl residues in the protein participated in the photochemical modification which resulted in the formation of new disulfide bonds between the cysteinyl residues and thiouri-dylic acid or its photoproducts. In addition, photooxidation at methionyl residues in the protein was suggested. The major portion of the aggregates was found to represent dimers on the basis of molecular weight, determinations by gel filtration and SDS-gel electrophoresis. They were fractionated into 3 fractions. (1) Aggregates dissociable by denaturation which were formed by non-covalent interactions. (2) Aggregates dissociable by reduction which were formed presumably by disulfide interchanges between the two monomer molecules. (3) Aggregates undissociable by both denaturation and reduction of which chemical natures are unknown.

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