A new radioactive cross-linking reagent for studying the interactions of proteins

We have developed a photoactivable, heterobifunctional, reversible, radioactively labeled, chemical cross-linking reagent, 3-[(2-nitro-4-azidophenyl)-2-aminoethyldithio]-N-succinimidyl propionate, for studying the interactions of proteins in situ. When reacted in the dark with a purified protein, it forms a covalent derivative which can be purified and reconstituted into biological systems. This derivative will form cross-links to neighboring macromolecules only upon photolysis; reduction cleaves the cross-link and transfers the radiolabel to the second molecule, which can then be identified by standard techniques. We have tested the cross-linker using the binding of gelatin to fibronectin. The cross-linker gives the proper chemical behavior under biological conditions, reacts with high yield and with a very low level of nonspecific cross-linking, and can be used to identify protein-protein and other interactions at the cell surface and elsewhere. The advantages, limitations and possible uses for this reagent are discussed.