Anion-exchange chromatographic behavior of recombinant rat cytochrome b5. Thermodynamic driving forces and temperature dependence of the stoichiometric displacement parameter Z

David J. Roush, Davinder S. Gill, Richard C. Willson

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

The HPLC anion-exchange isocratic retention behavior of the recombinant soluble core of wild type rat cytochrome b5 on Mono Q HR 5/5 was investigated as a function of temperature and sodium chloride concentration at fixed eluent flow-rates. Retention was measured over a range of eluent flow-rates at a specified temperature to determine if true adsorption equilibrium could be approximated by the HPLC method. Apparent Van't Hoff enthalpies of adsorption obtained from the HPLC retention data were positive, indicating an entropically driven spontaneous adsorption process, and were found to decline with increasing ionic strength. The retention results were interpreted in terms of the stoichiometric displacement model to obtain the apparent number of binding sites in the contact region, Z, as a function of temperature and of protein concentration. Z was found to depend significantly on temperature, even under conditions of nearly complete protein recovery, but did not depend on protein concentration at the low loadings studied.

Original languageEnglish (US)
Pages (from-to)207-218
Number of pages12
JournalJournal of Chromatography A
Volume653
Issue number2
DOIs
StatePublished - Nov 5 1993

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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