High-Performance Frontal Analysis of the Binding of Thyroxine Enantiomers to Human Serum Albumin Binding of Thyroxine Enantiomers to Human Serum Albumin Kimura

Abstract

The aim of this study was to characterize the binding property between thyroxine and human serum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system consisting of an HPFA column, an extraction column, and an analytical HPLC column was developed to be used to determine the unbound concentrations of thyroxine enantiomers. Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities. The binding constant (K) and the number of binding sites on an HSA molecule (n) evaluated from Scatchard plot analysis were K = 1.01 x 10(6)m(-1) and n = 1.90 for L: -thyroxine, and K = 9.71 x 10(5) m(-1) and n = 1.97 for D: -thyroxine. The binding sites were identified using phenylbutazone and diazepam as site-specific probes for sites I and II, respectively, and each enantiomer was found to bind to both sites. Incorporation of a chiral HPLC column into the on-line system permitted the investigation of enantiomer-enantiomer interactions, which revealed that both enantiomers competitively bind to the same binding sites without significant allosteric effects.