Screening major binding sites on human serum albumin by affinity capillary electrophoresis.

Hee Seung Kim, John Austin, David S. Hage

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)169-187
Number of pages19
JournalMethods in molecular biology (Clifton, N.J.)
Volume276
StatePublished - 2004

Fingerprint

Capillary Electrophoresis
Serum Albumin
Binding Sites
Buffers
Warfarin
2-Hydroxy-5-nitrobenzyl Bromide
Apazone
Tetranitromethane
Ibuprofen
Electrophoretic Mobility Shift Assay
Dextrans
Benzodiazepines
Phosphates
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Screening major binding sites on human serum albumin by affinity capillary electrophoresis. / Kim, Hee Seung; Austin, John; Hage, David S.

In: Methods in molecular biology (Clifton, N.J.), Vol. 276, 2004, p. 169-187.

Research output: Contribution to journalArticle

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