PURPOSE: 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). METHODS: 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. RESULTS: 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.
PURPOSE: The aim of this study was to characterize the binding property between thyroxine and humanserum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA). METHODS: 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. RESULTS: Both enantiomers were bound to humanserum 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.