Photosensitized Modification of Bovine Pancreatic Ribonuclease by a Colored Substrate Analogue 4-Thiouridylic Acid

Abstract

Bovine pancreatic ribonuclease [EC 2. 7. 7. 16] (RNase) was inactivated by irradia-tion with ultraviolet light of 330-365mμ in the presence of a substrate analogue, 4-thiouridine-2'(3')-phosphate (4-thiouridylic acid), while a little or no inactivation was found in absence of the nucleotide and in the presence of a nucleoside, 4-thiouridine. The inactivation was inhibited by an excess amount of uridylic acid. The inactivation was notable at pH 5.6 and was feeble at pH's 3.6 and 7.6, and the degree of the inactivation was in paralell with the degree of the interaction between the enzyme and the nucleotide. It was deduced from these results that the complexing between RNase and 4-thiouridylic acid is an essential step for the photosensitized inactivation of the enzyme. Oxygen was required for the inactivation.
A photoproduct of RNase irradiated in the presence of 4-thiouridylic acid was isolated. Chemical analyses of this product revealed that histidyl, phenylalanyl and valyl residues at the active site were intact after the irradiation, but one tyrosyl residue was modified. The spectrophotometric titration of the irradiated protein suggested that the native conformation was retained.