Site specific photocleavage of proteins

Bifunctional probes containing an amino acid or a short peptide attached to a hydrophobic pyrenyl moiety were designed for the purpose of inducing site specific photocleavage of proteins. The amino acid and the short peptide serve as a recognition element to seek out specific binding sites on the target proteins. Site specific photocleavage of bovine serum albumin (BSA), chicken egg-white lysozyme, and carboxypeptidase A by pyrenyl probes such as N-(L-phenylalanine)-4(1-pyrene)butyramide (Py-Phe) was achieved in the presence of an electron acceptor, Co(III) hexammine chloride (CoHA). Electrophoresis experiments indicated two new bands corresponding to lower molecular weights than the native protein when the mixture of the probe, protein, and CoHA was exposed to 344 nm irradiation. No cleavage was evident in the absence of either one of the reagents or light. Absorption, fluorescence, and circular dichroism studies indicated the strong binding interactions of the probes with the target proteins. N-terminal peptide sequencing of the fragments indicated a single cleavage site in the proteins. The cleavage of BSA by Py-Phe was found to be between Leu 346 and Arg 347, while the cleavage of lysozyme occurred between Trp 108 and Val 109.^ Substitution of CoHA with other Co(III) complexes did not alter the photocleavage specificity. The probe side chain has a strong effect on the binding site selectivity on the proteins. Selected biological recognition elements can be coupled to a photoreactive moiety to deliver reagents to the protein target sites. Photocleavage of BSA by affinity peptide probes with the structures of Py-Gly-X (X = Phe, Tyr, Trp, and His) and Py-(Gly)$\sb{n}$-Phe (n = 0, 1, 2) resulted in different cleavage patterns.^ Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a transient absorption peak at ${\sim}460$ nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of the pyrenyl chromophore by Co(III) complex followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein backbone cleavage.