Preparation and application of radioiodinated sulfhydryl reagents for the covalent labeling of SH-proteins present in minute quantities.
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, kappa 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, kappa 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 [125I]tyramine-MIB (and not the other intermediates) to small Sephadex G-10 column and its elution with ethanol. [125I]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/mumol), 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 Mr = 32,000, which is most likely an externally oriented surface plasma membrane SH-protein. [125I]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.
Hazum
E
E
Division of Endocrinology, Glaxo Research Laboratories, Research Triangle Park, NC 27709.
Shisheva
A
A
Shechter
Y
Y
D000273:Adipose Tissue / Q000737:chemistry; D000818:Animals; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000737:chemistry*; D051381:Rats; D013329:Structure-Activity Relationship; D013388:Succinimides; D013439:Sulfhydryl Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D014439:Tyramine
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013388:Succinimides; D013439:Sulfhydryl Reagents; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D014439:Tyramine