title: Determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction.
abstract: A method was created on the basis of ultrafast affinity extraction to determine both the dissociation rate constants and equilibrium constants for drug-protein interactions in solution. Human serum albumin (HSA), an important binding agent for many drugs in blood, was used as both a model soluble protein and as an immobilized binding agent in affinity microcolumns for the analysis of free drug fractions. Several drugs were examined that are known to bind to HSA. Various conditions to optimize in the use of ultrafast affinity extraction for equilibrium and kinetic studies were considered, and several approaches for these measurements were examined. The dissociation rate constants obtained for soluble HSA with each drug gave good agreement with previous rate constants reported for the same drugs or other solutes with comparable affinities for HSA. The equilibrium constants that were determined also showed good agreement with the literature. The results demonstrated that ultrafast affinity extraction could be used as a rapid approach to provide information on both the kinetics and thermodynamics of a drug-protein interaction in solution. This approach could be extended to other systems and should be valuable for high-throughput drug screening or biointeraction studies.
authors.affiliation: Department of Chemistry, University of Nebraska , 704 Hamilton Hall, Lincoln, Nebraska 68588, United States.
authors.forename: Xiwei
authors.initials: X
authors.lastname: Zheng
authors.forename: Zhao
authors.initials: Z
authors.lastname: Li
authors.forename: Maria I
authors.initials: MI
authors.lastname: Podariu
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D006801:Humans; D007700:Kinetics; D004364:Pharmaceutical Preparations / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D004364:Pharmaceutical Preparations; D012709:Serum Albumin