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

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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.