Radioiodinated photoactivatable sulfhydryl specific reagents for the study of transducin domains

Two new hetero-bifunctional, sulfhydryl reactive, radioiodinated, photoactivatable compounds, N,N'-di(iodoazidophenylpropionamido)cystamine S-monoxide (125IDACO) and N-(iodoazidophenylpropionamido)cysteinyl-2'-thiopyridine (125IACTP) have been synthesized. These compounds were used to derivatize bovine holo transducin (holo T) and the results analyzed by SDS-PAGE. Titration of holo T with 125IDACO in the dark showed the following: (1) two of the three SH groups of T alpha could be titrated; (2) a fast titration of 1 mole 125IDACO per mole of T alpha occurred; (3) approximately 2 moles of 125IDACO per mole of T beta reacted. Titration of holo T with 125IACTP in the dark showed the following: (1) A fast titration of 1 mole 125IACTP per mole of T alpha occurred; (2) approximately 0.5 to 1.0 mole of 125IACTP per mole of T beta reacted. Tryptic digestion of 125IACTP-holo T followed by analysis on SDS urea gels, in the absence of reducing agents, showed radiolabel in a 3 kDa fragment and a 12-14 kDa fragment. Upon photolysis, the 125I label was transferred to a fragment of approximately 12 kDa