The occurrence of O-acylation during biotinylation of gonadotropin-releasing hormone and analogs: Evidence for a reactive serine

Gonadotropin-releasing hormone (GnRH) and two of its analogs ([D-Lys⁶]GnRH and des-Gly¹⁰-[D-Trp⁶]-GnRH) were reacted with sulfonated N-hydroxysuccinimide esters of biotin that have been reported to react specifically with primary amino groups. Fractionation by reversed-phase high performance liquid chromatography demonstrated the occurrence of multiple biotinylated derivatives for each reacted peptide. These results were unexpected since GnRH and des-Gly¹⁰-[D-Trp⁶]GnRH contained no reactive amino groups and [D-Lys⁶]GnRH had only one. Reaction of the biotinylated derivatives with hydroxylamine indicated that significant O-biotinylation had occurred. Mass spectrometric analyses established the stoichiometry of biotinylation and confirmed that substantial O-biotinylation of residue Ser⁴, and to a minor extent Tyr⁵, of GnRH and the two analogs had occurred. In contrast, the biotinylation of selected peptides unrelated to GnRH under identical reaction conditions indicated no significant evidence of O-acylation of seryl residues. Strikingly, biotinylation of GnRH under denaturing conditions largely abolished O-acylation, indicating that the observed O-biotinylation was dependent on peptide conformation. All the O-biotinylated derivatives displayed significantly reduced bioactivity. Taken together, these results give strong evidence that the Ser⁴ hydroxyl of GnRH has a significantly elevated intrinsic reactivity, which raises new questions concerning its putative role in the conformation and mode of action of the hormone. These results also demonstrate for the first time that the N-hydroxysuccinimide-biotin esters are capable of significant O-acylation and may be generally useful reagents for detecting highly reactive hydroxyamino acid residues.