125I-Labeled Crosslinking Reagent That is Hydrophilic, Photoactivatable, and Cleavable through an Azo Linkage

A radioactive crosslinking reagent, N-[4-(p-azido-m-[125I]iodophenylazo)benzoyl]-3-aminopropyl-N′-oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with125I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an125I-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid using carrier-free Na125I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lower by a factor of 3.6 than that of the heavy chain. These results demonstrated the specificity of the derivatized protein A-Sepharose as a photoaffinity probe. Photolabeling of IgG was the result of nitrene-mediated reactions and was not due to the incorporation of free125I.