Preparation and application of radioiodinated sulfhydryl reagents for the covalent labeling of SH-proteins present in minute quantities

Abstract In this study we have searched for sulfhydryl reagents which can be radiolabeled and detect minute quantities of SH-proteins. Iodoacetamidotyramine reacts with sulfhydryls at a low rate, having a pseudo-first order rate constant, κ obs = 3±0.2 M −1 s −1 , at neutral pH. In contrast, N -ethylmaleimide-containing reagents, such as tyrosine-MIB and tyramine-MIB were three orders of magnitude more reactive in alkylating sulfhydryls. Pseudo-first order rate constants, κ obs , were in the range of 5200–5700 M −1 s −1 . Therefore, a simple and convenient procedure was designed for the synthesis and the radioactive labeling of tyramine-MIB. Simplification was attained by virtue of the specific-‘affinity’ adsorption of [ 125 I]tyramine-MIB (and not the other intermediates) to small Sephadex G-10 column and its elution with ethanol. [ 125 I]Tyramine-MIB was stable for weeks in dried form and for hours in acidic to neutral aqueous solutions. The reagent, when radiolabeled to high specific activity (0.5 Ci/μmol), detected sulfhydryl proteins at concentrations as low as 1–10 pM. The applicability of the reagent in studying biological systems was demonstrated by adding it to intact adipocytes and the consequent labeling of a single protein with an apparent M r = 32 000, which is most likely an externally oriented surface plasma membrane SH-protein. [ 125 I]Tyramine-MIB reactivity and sensitivity exceeds that of protein-tyrosyl radioiodination by the chloramine-T procedure and is expected to assist in studying minute quantities of SH-proteins.