Photochemical crosslinking of neighboring residues in protein-nucleic acid complexes: RNase and pyrimidine nucleotide inhibitors

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Abstract
Irradiation of the stable complexes formed between RNase and its competitive inhibitors cytidine 2''(3''),5''-diphosphate (pCp), and uridine 2''(3''),5''-diphosphate (pUp), resulted in covalent bond formation between the pyrimidine nucleotides and the enzyme. The photochemical reactions were initiated by UV light of .lambda. > 300 nm, employing acetone as a photosensitizer. This method yielded less undesired by-products, particularly photolyzed amino acids and aggregates resulting from protein-to-protein cross-linking, than the direct method of irradiation with light of .lambda. > 260 nm. Tryptic digestion of the modified protein, and chromatographic analysis of the peptides thus obtained, revealed a single and specific peptide which became covalently linked to both nucleotide inhibitors. The amino acid composition of this peptide is consistent with the sequence Asn-67-Arg-85 of RNase. Part of this peptide (residues 78 through 83) is close to the enzyme''s binding site for the pyrimidine moiety of the nucleotides. Denatured RNase failed to cross-link to the inhibitors, and the extent of pUp cross-linking could be reduced by the addition of another competitive inhibitor (3''-UMP). The presence of excess inhibitor in the irradiation mixture did not lead to nonspecific cross-linking. This indicates that specificity is also achieved due to the fact that unbound excited inhibitor molecules do not react with the protein but prefer to follow different and faster reaction paths.