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.
Rissler
K
K
University of Freiburg, Department of Neurology, Germany.
Cramer
H
H
Engelmann
P
P
D000818:Animals; D001921:Brain / Q000378:metabolism; D002700:Chloramines / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007457:Iodine Radioisotopes / Q000737:chemistry*; D008297:Male; D008623:Mercaptoethanol / Q000737:chemistry; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014105:Tosyl Compounds / Q000737:chemistry*
D002700:Chloramines; D007457:Iodine Radioisotopes; D018040:Receptors, Neurokinin-1; D014105:Tosyl Compounds; C016300:chloramine-T; D013373:Substance P; C055640:substance P, Tyr(8)-; D008623:Mercaptoethanol