title: Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta-bromoacetoxydihydrotestosterone.
abstract: This article deals with the elucidation of the steroid-binding site of human sex hormone-binding globulin (SHBG). 17 beta-Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in a time-dependent and irreversible fashion. The interaction could be totally inhibited by the simultaneous addition of an excess of dihydrotesterone. At the completion of the reaction, the molar ratio of BA-DHT to SHBG was approximately unity. SHBG was affinity labeled with [14C]BA-DHT and submitted to acid hydrolysis. The released amino acids were evaluated on high performance liquid chromatography, and virtually all of the 14C was identified as 3-[14C]carboxymethylhistidine. Furthermore, [14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation of the released tryptic peptides by reverse-phase high performance liquid chromatography. The 14C was localized to a single tryptic peptide. It contained 2' histidyl residues, corresponding to residues 235 and 251 in the known amino acid sequence of SHBG. Although most of the 3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was trapped on the filter of the amino acid sequenator, sufficient radioactivity emerged to identify histidyl residue 235 as the labeled amino acid.
authors.affiliation: Department of Medicine, St. Luke's/Roosevelt Hospital Center, New York, New York 10019.
authors.forename: M S
authors.initials: MS
authors.lastname: Khan
authors.forename: W
authors.initials: W
authors.lastname: Rosner
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005260:Female; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011247:Pregnancy; D012738:Sex Hormone-Binding Globulin / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
chemical_list: D000345:Affinity Labels; D010446:Peptide Fragments; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; D006639:Histidine; C023562:dihydrotestosterone 17-bromoacetate; D014357:Trypsin