Effect of mobile phase composition on the binding kinetics of chiral solutes on a protein-based high-performance liquid chromatography column: interactions of D- and L-tryptophan with immobilized human serum albumin.
This work examined the kinetic interactions of chiral solutes on immobilized protein columns, using the binding of D- and L-tryptophan to human serum albumin as a model. Based on band-broadening studies and previous measurements of the association equilibrium constants (Ka) for this system, estimates were obtained for the dissociation and association rate constants (kd and ka) for D- and L-tryptophan under a variety of operating conditions. The relative importance of ka versus kd in creating changes in the overall binding affinity was then considered. For example, an increase in temperature from 4 to 45 degrees C gave a large change in ka for L-tryptophan that was due both to an increase in kd and to a decrease in ka, while ka and kd for D-tryptophan showed a parallel increase that led to a much smaller temperature dependence for Ka. Similar comparisons between ka, kd and Ka were performed over a range of pH values, ionic strengths and solvent polarities. It was also possible from these studies to examine the changes in enthalpy and entropy that accompanied the formation of the activated complex between human serum albumin and each solute. The results from this work were then used to illustrate the importance of kinetics and band-broadening in protein-based chiral separations, and an example was provided showing how this type of kinetic data might be used to help optimize such separations.
Yang
J
J
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Hage
D S
DS
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry*; D012996:Solutions; D013237:Stereoisomerism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
D007202:Indicators and Reagents; D012709:Serum Albumin; D012996:Solutions; D014364:Tryptophan