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Interactions in affinity partition studied using fluorescence spectroscopy.

P A Alred1, G Johansson, F Tjerneld

  • 1Department of Biochemistry, University of Lund, Sweden.

Analytical Biochemistry
|September 1, 1992
PubMed
Summary
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This study quantifies the binding of Procion Yellow HE-3G dye to lactate dehydrogenase using fluorescence titration. The method accurately measures protein-ligand interactions in polymer solutions and aqueous two-phase systems.

Area of Science:

  • Biochemistry
  • Protein-ligand interactions
  • Fluorescence spectroscopy

Background:

  • Lactate dehydrogenase (LDH) is a crucial enzyme in cellular metabolism.
  • Triazine dyes are widely used in protein purification and biochemical studies.
  • Understanding protein-dye interactions is essential for optimizing purification processes.

Purpose of the Study:

  • To determine the binding constant and number of binding sites for Procion Yellow HE-3G to rabbit muscle lactate dehydrogenase.
  • To evaluate the utility of fluorescence titration for characterizing protein-ligand interactions in various environments, including polymer solutions and aqueous two-phase systems.
  • To assess the applicability of these measurements in affinity-based protein purification strategies.

Main Methods:

Related Experiment Videos

  • Fluorescence titration was employed to measure the binding of Procion Yellow HE-3G to lactate dehydrogenase.
  • Experiments were conducted with the dye free in solution or conjugated to polymer carriers (polyethylene glycol and dextran).
  • Binding studies were performed in buffer solutions, polymer solutions, and aqueous two-phase systems (PEG-dextran).

    • Main Results:

    • The binding constant and number of binding sites for Procion Yellow HE-3G to lactate dehydrogenase were successfully determined.
    • Measurements in aqueous two-phase systems showed good agreement between experimentally determined and theoretically calculated partition coefficients for lactate dehydrogenase using PEG-Procion Yellow as a ligand.
    • Fluorescence titration provided reliable binding data in the complex environments relevant to protein purification.

    Conclusions:

    • Fluorescence titration is a valuable technique for quantifying protein-ligand interactions, particularly for polymer-conjugated ligands.
    • The binding characteristics determined by fluorescence titration correlate well with affinity partition behavior in aqueous two-phase systems.
    • This method offers an advantage by allowing binding strength measurements in the actual environment of protein-ligand complex formation during purification.