Photoreactive Biotinylated Peptide Ligands for Affinity Labeling

Summary

The combination of photoaffinity labeling with reversible biotin-avidin complex formation, a method termed PAMAC (photoaffinity-mediated avidin complexing), is a powerful approach in the analysis of ligand-receptor systems that combines the advantages of both techniques. Ligands are designed either for permanent or temporary biotinylation and are — if necessary after additional radiolabeling — photocross-linked to receptors. Biotinylated irreversible conjugates or subsequent enzymatic fragments can be isolated by avidin-streptavidin affinity chromatography. Temporary biotinylation allows isolation of covalent ligand-receptor complexes after cleavage of the biotin group. With permanent biotinylation, all preparative and analytical advantages of the biotin label are preserved, for instance, high-sensivity detection by enhanced chemiluminescence, or dot-staining procedures.

Using insulin as an example, practical procedures for synthesis of a trifunctional labeling reagent, 2 photo/biotin-labeled hormone derivatives, and their applications in receptor binding-site analysis are described in detail. Brief reference is made to related work with other peptide hormones.