Photoreactive insulin derivatives for the detection of the doubly labeled insulin receptor

Two different photoinsulins, the radioactive N(epsilonB29)-(4-azidosalicyloyl) insulin and the novel biotinylated N(epsilonB29)-(4-azidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to study the binding stoichiometry of insulin and the insulin receptor in a direct approach. Both derivatives were cross-linked simultaneously to the (alphabeta)(2) receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes were isolated by streptavidin-affinity chromatography. Analysis of both markers, the radioactive (125)I marker and the biotin marker, proved the existence of a covalent complex of one receptor molecule and two ligands. Thus, orthogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes.