Preferred high-performance liquid chromatographic anion-exchange chromatographic contact region for recombinant rat cytochrome b5

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

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

The HPLC anion-exchange isocratic retention behaviors of site-directed charge mutants of the recombinant soluble core of rat cytochrome b5 on Mono Q HR 5 5 were investigated as a function of sodium chloride concentration, at a fixed temperature and eluent flow-rate. The retention behavior of the charge mutants was observed to depend on both the net charge of the protein and on the distribution of charged residues on the protein surface. Site-directed mutants of the same net charge differed significantly in retention behavior; differences in retention were observed to increase with decreasing ionic strength. The retention results were interpreted in terms of the stoichiometric displacement model (SDM) to obtain the apparent number of binding sites in the contact region, Z. Elimination of a single negatively charged residue, in an apparent preferred chromatographic contact region, resulted in disproportionately large changes in the retention behavior as compared with elimination of a single negatively charged residue on other areas of the protein surface. The experimentally determined preferred chromatographic contact region compares favorably with the results of batch equilibrium adsorption studies on the same system and with previously reported results of computational molecular electrostatic modeling. The results of this study indicate that the observed protein ion-exchange retention represents the summation of many fractional electrostatic interactions of varying interaction energies. Therefore, protein ion-exchange retention is a function of both the number of contacts and the individual electrostatic interaction energies of the contacts between the protein and the ion-exchange surface.

Original languageEnglish (US)
Pages (from-to)339-349
Number of pages11
JournalJournal of Chromatography A
Volume704
Issue number2
DOIs
StatePublished - Jun 9 1995

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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