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

Two different photoinsulins, the radioactive N-is an element of B29-(4-azid osalicyloyl) insulin and the novel biotinylated N-is an element of B29-(4-a zidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to st udy the: binding stoichiometry of insulin and the insulin receptor in a dir ect approach. Both derivatives were cross-linked simultaneously to the (alp ha beta)(2) receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes w ere isolated by streptavidin-affinity chromatography. Analysis of both mark ers, the radioactive I-125 marker and the biotin marker, proved the existen ce of a covalent complex of one receptor molecule and two ligands. Thus, or thogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes. (C) 2000 Elsevier Science Inc. Al l rights reserved.