Bioanalytical study of the effect of protein variants upon biological activity of endogenous compounds

建立了由高效前沿分析柱、萃取柱和分析柱组成的在线HPLC系统，用于测定甲状腺素对映体的游离浓度。两种对映异构体在两个具有相似亲和力的高亲和力位点与人血清白蛋白结合，并与位点I和位点II结合。将手性HPLC柱与在线系统联用，研究了对映体-对映体相互作用，发现两种对映体竞争性结合在同一结合位点上，无明显的变构效应，该系统与电化学检测器联用，可同时测定未结合的甲状腺激素人血浆中T_4、T_3和rT_3的测定结果与传统EIA法一致。该系统允许直接、同时和非RI测定，这是常规方法的独特特征，并且适用于蛋白质变体的结合分析。

An on-line HPLC system consisting of an high-performance frontal analysis (HPFA) column, an extraction column and an analytical HPLC column was developed to determine the unbound concentrations of thyroxine enantiomers. Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities, and were bound to both site I and site II. Incorporation of a chiral HPLC column into the on-line system allowed the investigation of enantiomer-enantiomer interaction, which revealed that both enantiomers are bound to the same binding sites competitively without any significant allosteric effect.This system was coupled with electrochemical detection (ECD) for the simultaneous determination of unbound thyroid hormones (T4,T3 and rT3) in human plasma A few pM levels of the unbound concentrations can be determined with good reproducibility, and the results agreed with the convensional EIA method. This system allows the direct, simultaneous and non-RI assay which is the unique feature over conventional methods, and is applicable to the binding analysis of protein variants.