title: Screening major binding sites on human serum albumin by affinity capillary electrophoresis.
abstract: A screening method is described for determining whether a drug or small solute has significant interactions at the two major binding sites on human serum albumin (HSA). This method uses affinity capillary electrophoresis (ACE) to perform a mobility shift assay, where the solute of interest is injected in both the presence of pH 7.4, 0.067 M phosphate buffer, and the same buffer containing a known concentration of HSA. Dextran is also used in the running buffer to adjust the mobility of HSA. Two types of modified HSA are used in this assay. The first is modified with 2-hydroxy-5-nitrobenzyl bromide (HNB), which selectively blocks HSA's warfarin-azapropazone site. The second type of HSA is modified with tetranitromethane (TNM), which decreases binding at the indole-benzodiazepine site. By comparing the mobility of a solute in the presence of these two modified forms of HSA vs normal HSA, it is possible to detect solute interactions at these binding sites. This approach is illustrated using warfarin and ibuprofen as examples of test solutes.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, NE., USA.
authors.forename: Hee Seung
authors.initials: HS
authors.lastname: Kim
authors.forename: John
authors.initials: J
authors.lastname: Austin
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000737:chemistry*; D001032:Apazone / Q000737:chemistry; D001569:Benzodiazepines / Q000737:chemistry; D019075:Electrophoresis, Capillary; D006801:Humans; D007052:Ibuprofen / Q000737:chemistry; D007211:Indoles / Q000737:chemistry*; D011485:Protein Binding; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry*; D013774:Tetranitromethane / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*; D014859:Warfarin / Q000737:chemistry*
chemical_list: D007211:Indoles; D012709:Serum Albumin; D001569:Benzodiazepines; D014859:Warfarin; D015077:2-Hydroxy-5-nitrobenzyl Bromide; C030374:indole; D014364:Tryptophan; D013774:Tetranitromethane; D001032:Apazone; D007052:Ibuprofen