Photoaffinity labeling of human sex hormone-binding globulin using 17 alpha-alkylamine derivatives of 3 beta-androstanediol substituted with azidonitrophenylamido, azidonitrophenylamino, or trifluoroazidonitrophenylamino chromophores. Localization of

摘要

Purified human SHBG was photoaffinity labeled with 17 alpha -aminomethyl (M), 17 alpha -aminoethyl (E), and 17 alpha -aminopropyl (P) derivatives of [3 alpha-H-3]-5 alpha -androstane-3 beta ,17 beta -diol coupled to 5-azido-2-nitrobenzoylamido (ANB), 4-azido-2-nitrophenylamino (ANP), and 5-azido-2-nitro-3,4,6-trifluorophenylamino (ANTFP) chromophores. Successful labeling was achieved in all cases except for the two photoreagents with the shortest side chains namely, ANP-M and ANTFP-M derivatives. Edman sequencing and mass spectrometry of immunopurified photolabeled tryptic fragments revealed that radioactivity was present either on the sequence of residues 73-94, uniquely at the level of Trp-84 (stable covalent labeling), or on one of the two overlapping sequences of residues 126-134 and 126-135, at the level of Pro-130 (labile labeling) and Lys-134 (either stable or partially labile labeling), respectively. The same Trp-84 was photolabeled with the three ANB derivatives of increasing lengths, and by the ANP-P photoreagent. This residue was the exclusive target for the shortest [H-3]ANB-M photoreagent but was a minor site for the longest [H-3]ANB-P photoreagent, essentially recovered at the level of Pro-130. The [H-3]ANB-E photoreagent of intermediate size also labeled exclusively Trp-84, except in some experiments in which photolabeling was recovered predominantly at the level of Pro-130. The [H-3]ANP-P photoreagent with an overall length similar to that of the ANB-P photoreagent labeled simultaneously Trp-84 (minor site) and Lys-134. The other [H-3]ANP-E, [H-3]ANTFP-E, and [H-3]ANTFP-P derivatives labeled in all cases Lys-134. These findings indicate that the conserved Trp-84 and the two Pro-130 and Lys-134 residues are all located in the vicinity of the D ring of steroid ligands and remain freely accessible from the C17 alpha position, thus providing biochemical data delineating the corresponding region of the steroid-binding site.