Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates.
This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions.
Yoo
Michelle J
MJ
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
Hage
David S
DS
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry*
D055767:Immobilized Proteins; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D012822:Silicon Dioxide