A new photoactivatable reagent capable of transferring a radiolabel to target proteins. Application to the human growth hormone-rat liver prolactin receptor interaction.

Abstract

A new photoactivatable cross-linking reagent, 1-(2'-dithiopyridyl)-2-(5'-azidosalicylamido)ethane (ASDPE), was synthesized. This probe can be easily labeled with 125I in the azidosalicylamido ring and contains an activated disulfide bridge. After reaction of [125I]ASDPE with proteins, the radiolabeled moiety of the probe becomes attached to cysteine residues. Upon partial reduction of human growth hormone (hGH) with dithiothreitol, its C-terminal disulfide bond between residues 182 and 189 was cleaved and the nascent thiol groups were modified with [125I]ASDPE to yield [125I]ASET-hGH [1-(thio-hGH)-2-(3'-[125I]iodo-5'-azidosalicylamido)ethane]. After binding of this hormone derivative to rat liver microsomes, followed by photolysis and subsequent reduction of disulfide bridges, the specific transfer of the radiolabeled moiety to prolactin receptor (PRL-R) was achieved. Partial purification of the radiolabeled receptor by size exclusion chromatography was performed. We anticipate that [125I]ASDPE will be generally useful in pursuing structural and functional studies of target proteins which interact specifically with protein ligands.