Fluorescent photoaffinity labeling: Adenosine 3′,5′-cyclic monophosphate receptor sites

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Abstract
An approach to the study of protein receptor sites in protein mixtures or supramolecular assemblies by using fluorescence spectroscopy is described. This approach, fluorescent photoaffinity labeling, combines the merits of photoaffinity labeling to attain site-directed reactivity with the probing power of fluorescent ligands. A fluorescent photoaffinity label for cyclic[c]AMP receptor sites of cAMP-dependent protein kinases was synthesized in both unlabeled and radioactive forms. The probe, 8-azido-1,N6-ethenoadenosine 3'',5''-cyclic monophosphate, mimics cAMP in its ability to stimulate the phosphotransferase activity of the protein kinases and strongly competes with cAMP for its binding sites in all preparations so far tested. Photolysis, after equilibration of protein kinase and 8-azido-1,N6-ethenoadenosine 3'',5''-cyclic monophosphate in the dark, effects binding of the intermediate nitrene irreversibly and specifically to the cAMP sites with the development of fluorescence. Excess reagent and low MW photolytic products are removable by dialysis. Studies of a crude beef heart preparation containing cAMP-dependent protein kinase suggest that the cAMP binding sites are hydrophobic in nature and strongly immobilize the adenine moiety of the cyclic nucleotide.