Application of [(125)I]-[Tyr8]-substance P prepared by the chloramine-T method to receptor-binding experiments after subsequent reduction with mercaptoethanol and purification by reversed-phase liquid chromatography.

Radiolabeling of [Tyr8]-substance P ([Tyr8]-SP) with the (125)I-isotope was performed by use of the chloramine-T technique. The primary formed radiolabeled product, having been quantitatively converted to the corresponding sulfoxide yielding [(125)I]-[Tyr8]-(Met11-->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 Met11 thioether structure was observed. The reduced peptide, consisting of [(125)I]-[Tyr8]-(Met11)-SP was separated from its by-products by reversed-phase high-performance liquid chromatography on octadecylsilyl silica gel with 100 mM 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 [Tyr8]-SP starting material and exhibited a specific activity of 1.9-10(6) Ci/M. In contrast to [(125)I]-[Tyr8]-->(Met11-->O)-SP, satisfactory receptor-binding was now observed with the [(125)I]-->[Tyr8]-(Met11)-SP derivative.