title: Characterization of the binding and chiral separation of D- and L-tryptophan on a high-performance immobilized human serum albumin column.
abstract: High-performance affinity chromatography was used to study the separation and binding of D- and L-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both D- and L-tryptophan were binding to single but distinct sites on HSA. L-Tryptophan bound to the indole site of HSA. D-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of D- and L-tryptophan at pH 7.4 and 25 degrees C were 0.4 x 10(4) and 2.7 x 10(4) M-1, respectively. The value of delta G for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of D- and L-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of D- and L-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln 68588-0304.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: D S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D001667:Binding, Competitive; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007211:Indoles / Q000737:chemistry; D010710:Phosphates; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry; D013237:Stereoisomerism; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000097:blood / Q000302:isolation & purification*; D014859:Warfarin / Q000097:blood
chemical_list: D007202:Indicators and Reagents; D007211:Indoles; D010710:Phosphates; D012709:Serum Albumin; D014859:Warfarin; D014364:Tryptophan