Photoaffinity Labeling of Human Sex Hormone-Binding Globulin Using 17α-Alkylamine Derivatives of 3β-Androstanediol Substituted with Azidonitrophenylamido, Azidonitrophenylamino, or Trifluoroazidonitrophenylamino Chromophores. Localization of Trp-84 in the Vicinity of the Steroid-Binding Site

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Abstract
Purified human SHBG was photoaffinity labeled with 17α-aminomethyl (M), 17α-aminoethyl (E), and 17α-aminopropyl (P) derivatives of [3α-3H]-5α-androstane-3β,17β-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 [3H]ANB-M photoreagent but was a minor site for the longest [3H]ANB-P photoreagent, essentially recovered at the level of Pro-130. The [3H]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 [3H]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 [3H]ANP-E, [3H]ANTFP-E, and [3H]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 C17α position, thus providing biochemical data delineating the corresponding region of the steroid-binding site.