Microcalorimetric studies of the interaction mechanisms between proteins and Q-Sepharose at pH near the isoelectric point (pI): Effects of NaCl concentration, pH value, and temperature

Fu Yung Lin, Chin Sung Chen, Wen Yih Chen, Shuichi Yamamoto

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77 Scopus citations

Abstract

This study examined the interaction mechanisms of β-lactoglobulins A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near the isoelectric point at which the proteins are expected to be electrically neutralized under various NaCl concentrations and temperatures by the equilibrium binding analysis and the adsorption enthalpy directly measured by isothermal titration calorimetry. The data evaluated from isotherms fitted by the Langmuirean model reveal that the addition of NaCl considerably reduced the binding affinities and capacities of both the proteins with Q-Sepharose at pH 5.2, indicating that electrostatic forces are dominant during the adsorption. However, the hydrophobic interaction seems to be involved in adsorption as well at a higher NaCl concentration, and the adsorption enthalpies confirm this suggestion. In addition, the effects of temperature on the equilibrium binding behaviors for Lg A or Lg B with Q-Sepharose were found to be salt concentration-dependent, probably due to their different binding mechanisms at 0.03 M and 0.3 M NaCl. Where, at 0.3 M NaCl, the hydrophobic interaction plays a more pronounced role. This implication was again supported by the adsorption enthalpies. The presented data provide further insight to the interaction mechanisms between proteins and ion exchangers, facilitating the optimization of protein separations.

Original languageEnglish
Pages (from-to)281-289
Number of pages9
JournalJournal of Chromatography A
Volume912
Issue number2
DOIs
StatePublished - 6 Apr 2001

Keywords

  • Adsorption
  • Calorimetry
  • Isothermal titration calorimetry
  • Lactoglobulins
  • Proteins
  • Thermodynamic parameters

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