Photocleavage of avidin by a new pyrenyl probe

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py–biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py–biotin were demonstrated. Binding of Py–biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py–biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342 nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

A new small reagent, d-biotinyl-1(1-pyrene)methylamide (Py–biotin), was designed to bind to specific site of avidin. Site-specific photocleavage of avidin was successfully achieved upon irradiation of avidin/Py–biotin solution in the presence of an electron acceptor, CoHA.Figure optionsDownload as PowerPoint slideHighlights
► Avidin was successfully cleaved at specific binding site by the new probe, Py–biotin.
► Two new cleaved fragments were obtained with cleavage site between Thr 77–Val 78.
► Photocleavage cannot be achieved in the absence of either electron acceptor, Py–biotin or light.