Chiral separation mechanisms in protein-based HPLC columns. 2. Kinetic studies of (R)- and (S)-warfarin binding to immobilized human serum albumin.
This work used plate height measurements to investigate the kinetics of (R)- and (S)-warfarin binding to an immobilized HSA column. The dissociation rate constants for (R)- and (S)-warfarin on this column increased from 0.06 to 1.9 s-1 and from 0.06 to 0.36 s-1 between 4 and 45 degrees C. The corresponding association rate constants increased from 2.4 x 10(4) to 3.2 x 10(5) M-1 s-1 for (R)-warfarin and from 4.4 x 10(4) to 7.2 x 10(4) M-1 s-1 for (S)-warfarin over the same temperature range. From the dissociation data, it was found that an increase in temperature led to a large decrease in the plate height due to stationary phase mass transfer for both enantiomers. Further studies indicated that (R)- and (S)-warfarin had similar activation energies for their binding to HSA. For (R)-warfarin, most of this energy requirement was due to the change in enthalpy of the system, while for (S)-warfarin, it was mainly due to the change in entropy. All of these results agree with an earlier model, in which (R)- and (S)-warfarin were proposed to interact with regions on the interior and exterior of HSA, respectively. In addition, these results offer a number of useful insights into the mechanisms of protein-based chiral separations.
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Department of Chemistry, University of Nebraska, Lincoln 68588, USA.
Hage
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D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007700:Kinetics; D012709:Serum Albumin / Q000378:metabolism*; D013237:Stereoisomerism; D014859:Warfarin / Q000302:isolation & purification* / Q000378:metabolism*
D012709:Serum Albumin; D014859:Warfarin