Application of [ 125I]-[Tyr 8]-substance P prepared by the chloramine-T method to receptor-binding experiments after subsequent reduction with mercaptoethanol and purification by reversed-phase liquid chromatography

Abstract

Radiolabeling of [Tyr 8]-substance P ([Tyr 8]-SP) with the 125I-isotope was performed by use of the chloramine-T technique. The primary formed radiolabeled product, having been quantitatively converted to the corresponding sulfoxide yielding [ 125I]-[Tyr 8]-(Met 11 → O)-Sp completely lacked any binding to proteins rich in SP receptor populations. However, after reductive treatment with mercaptoethanol for about 2 h, a complete reconstitution of the Met 11 thioether structure was observed. The reduced peptide, consisting of [ 125I]-[Tyr 8]-(Met 11)-SP was separated from its by-products by reversed-phase high-performance liquid chromatography on octadecylsilyl silica gel with 100 m M triethyl ammonium formate buffer containing 22% acetonitrile (pH 2.2). The labeled SP derivative prepared by this two-step synthesis was obtained in 73% overall yield related to the [Tyr 8]-SP starting material and exhibited a specific activity of 1.9·10 6 Ci/M. In contrast to [ 125I]-[Tyr 8]-(Met 11 →O)-SP, satisfactory recepto-binding was now observed with the [ 125I]-[Tyr 8]-(Met 11)-SP derivative.