PHOTOCHEMICAL PROTEASE - SITE-SPECIFIC PHOTOCLEAVAGE OF HEN EGG LYSOZYME AND BOVINE SERUM-ALBUMIN

Site-specific photocleavage of hen egg lysozyme and bovine serum album in (BSA) by N-(1-phenylalanine)-4-(l-pyrene)butyramide (Py-Phe) is rep orted. Py-Phe binds to lysozyme and BSA with binding constants 2.2+/- 0.3 x 10(5) M-1 and 6.5 +/- 0.4 x 10(7) M-1, respectively. Photocleava ge of lysozyme and BSA was achieved with high specificity when a mixtu re of protein, Py Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleava ge was ob served in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site o f lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA wa s found to be between Leu-346 and Arg-347. Laser flash photolysis stud ies of a mixture of protein, Py-Phe, and CoHA showed a strong transien t with absorption centered at approximate to 460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Ph e by CoHA followed by the reaction of the resulting pyrenyl cation rad ical with the protein backbone may be responsible for the protein clea vage. The high specificity of photocleavage may be valuable in targeti ng specific sites of proteins with small molecules.