Effects of Ligand Heterogeneity in the Characterization of Affinity Columns by Frontal Analysis

Stacey A. Tweed, Bounthon Loun, David S. Hage

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

This study examined how the binding capacities and equilibrium constants measured by frontal analysis are affected by ligand heterogeneity in affinity columns. Equations derived for n- and two-site systems gave good agreement with results obtained for the binding of L-thyroxine to a column containing human serum albumin (HSA) and for the binding of (R)-warfarin to coupled columns containing HSA or pigeon serum albumin. The same equations were used to examine how different degrees of ligand heterogeneity affected the apparent binding capacities or equilibrium constants measured using the linear range of double-reciprocal frontal analysis plots. A large proportion of two-site systems gave good estimates (i.e., less than 10-20% error) for the true total column capacity and for the association constant of the highest affinity ligand in the column. A smaller, but still appreciable, fraction of all three- and four-site cases also produced good estimates of these values. The results of this work are not limited to protein-based affinity columns but should be applicable to any type of stationary phase that has well-defined binding regions and relatively fast, reversible interactions with solutes.

Original languageEnglish (US)
Pages (from-to)4790-4798
Number of pages9
JournalAnalytical chemistry
Volume69
Issue number23
DOIs
StatePublished - Dec 1 1997

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Serum Albumin
Equilibrium constants
Ligands
Warfarin
Thyroxine
Association reactions
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Effects of Ligand Heterogeneity in the Characterization of Affinity Columns by Frontal Analysis. / Tweed, Stacey A.; Loun, Bounthon; Hage, David S.

In: Analytical chemistry, Vol. 69, No. 23, 01.12.1997, p. 4790-4798.

Research output: Contribution to journalArticle

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