Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives.
A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.
Kim
Hee Seung
HS
Department of Chemistry, University of Nebraska, Lincoln, NE 68588-0304, USA.
Austin
John
J
Hage
David S
DS
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000737:chemistry; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry; D001665:Binding Sites; D002021:Buffers; D002619:Chemical Warfare Agents / Q000737:chemistry; D003911:Dextrans; D019075:Electrophoresis, Capillary / Q000379:methods*; D005480:Flurbiprofen / Q000737:chemistry; D006801:Humans; D007052:Ibuprofen / Q000737:chemistry; D015394:Molecular Structure; D012709:Serum Albumin / Q000032:analysis*; D013496:Suprofen / Q000737:chemistry; D013774:Tetranitromethane / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014859:Warfarin / Q000737:chemistry
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002021:Buffers; D002619:Chemical Warfare Agents; D003911:Dextrans; D012709:Serum Albumin; D005480:Flurbiprofen; D014859:Warfarin; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D013496:Suprofen; D013774:Tetranitromethane; D007052:Ibuprofen