Site-specific photocleavage of lysozyme by N-4( 1-pyrene)butyroyl-L-phenylalanine (Py-Phe) prompted us to investigate the role of the peptidyl side chain in determining the specificity of the protein photocleavage. The spectroscopic and photocleavage properties of Py-Gly, Py-(Gly)(n)-Phe (n = 0, 1, 2), and Py-Phe-Gly-Gly, in this context, is described here. The linker length/structure of these molecules has a profound effect on the spectroscopic and photocleavage properties of the probes. The absorption spectral changes accompanying the binding of the probes Py-(Gly)(n)-Phe to the proteins were independent of the linker structure or length. Binding constants of these probes with proteins such as bovine serum albumin (BSA) or lysozyme, varied from 2.2. +/- 0.3 x 10(5) dm(3)mol(-1) to 6.5 +/- 0.4 x 10(7) dm(3)mol(-1). Binding constants of Py-Phe and Py-Phe-Gly-Gly for BSA have been an order of magnitude larger than those of Py-Gly, Py-Gly-Phe or Py-Gly-Gly-Phe. The fluorescence spectral changes, in contrast to the absorption changes, depended on the probe structure suggesting the subtle role of the linker structure on the probe binding properties. In addition to the pyrenyl fluorescence, new, broad emission was observed at 466 nm with Py-Phe and Py-Phe-Gly-Gly when bound to BSA, but no such emission was observed with the other probes or with any of these probes bound to lysozyme. Much clearer distinction between the probes can be ascertained from the circular dichroism (CD) spectra. No two CD spectra of the probe-protein complexes were superimposable, clearly demonstrating the differences in the chiral environment surrounding the pyrenyl chromophore in the protein matrix. Fluorescence quenching experiments using Co(III)hexammine (CoHA) as the quencher indicate extensive protection of the fluorophore bound to BSA, while the chromophore bound to lysozyme was relatively more accessible. The tripeptide probes were protected better than the dipeptide probes. The distance of separation between the carboxyl function and the pyrenyl group in these probes has a substantial effect on the accessibility of the probe to CoHA. These differences are, in turn, expected to influence the photocleavage efficiencies. Photocleavage of lysozyme was observed when probe-protein complexes have been irradiated at 344 nm in the presence of CoHA as an electron acceptor. All of the probes showed high specificity with lysozyme and resulted in just two product bands. In case of BSA, the protein cleavage site location and specificity varied drastically with the probe structure, suggesting major changes in the selectivity as a function of probe structure. Peptide sequencing studies of the photofragments from lysozyme revealed the location of the photocleavage sites. While Py-(Gly),-Phe (n = 1, 2) cleave lysozyme at a site between Trp108 and Val109, similar to that for Py-Phe, a second minor cleavage site at Ala110/Trp111 was observed for Py-(Gly)(n)-Phe (0, 1, 2) and Py-Phe-Gly-Gly. The ratio of the yields of the major to the minor product, with lysozyme, depended on the nature of the linker. No protein cleavage was observed in the absence of the probe or CoHA or light. The spectral and the photochemical studies indicate the binding and cleavage specificity of the probes vary with the side chain structure.