Competitive ion-exchange adsorption of proteins: Competitive isotherms with controlled competitor concentration

Tony Cano, Natalie D. Offringa, Richard C. Willson

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

The competitive adsorption processes inevitably present in chromatographic separations of complex mixtures have not been extensively studied. This is partly due to the difficulty of measuring true competitive isotherms, in which all system parameters (including competitor concentrations) are held constant. We report a novel approach to determining competitive protein adsorption isotherms in which the competitor concentration is held constant across the entire isotherm. By using the heme prosthetic group in cytochrome b5 as a quantitative spectrophotometric label, competitive isotherms between cytochrome b5 and α-lactalbumin can be constructed. Similarly, manganese-substituted protoporphyrin IX heme replacement allows the non-perturbing labeling of individual cytochrome b5 conservative surface charge mutants by replacement of a single atom in the interior of the protein. This labeling allows the study of competition between cytochrome b 5 charge mutants of identical size and shape, which differ only in charge arrangement. Using these techniques, the effect of competing species on equilibrium behavior and the apparent heterogeneity of anion-exchange adsorbents in the presence of competitors can be quantitatively studied by fitting the data to two popular single-component binding models, the Temkin and the Langmuir-Freundlich (L-F) isotherms.

Original languageEnglish (US)
Pages (from-to)116-126
Number of pages11
JournalJournal of Chromatography A
Volume1079
Issue number1-2 SPEC. ISS.
DOIs
StatePublished - Jun 24 2005

Keywords

  • Anion-exchange
  • Competitive adsorption
  • Competitive binding
  • Competitive isotherms
  • Ion-exchange
  • Langmuir-Freundlich isotherm
  • Protein adsorption
  • Temkin isotherm

ASJC Scopus subject areas

  • Analytical Chemistry

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