Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling.
The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus nonretained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (+/-0.2) s(-1) was obtained by this approach at pH 7.4 and 37 degrees C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates.
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
John E
JE
Schiel
Corey M
CM
Ohnmacht
David S
DS
Hage
D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D012709:Serum Albumin / Q000032:analysis / Q000378:metabolism; D014364:Tryptophan / Q000032:analysis / Q000378:metabolism
D004364:Pharmaceutical Preparations; D011506:Proteins; D012709:Serum Albumin; D014364:Tryptophan