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Related Experiment Videos

Protonation effect on drug affinity.

Robert B Raffa1, Gregory W Stagliano, Shawn D Spencer

  • 1Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA 19140, USA. robert.raffa@temple.edu

European Journal of Pharmacology
|January 20, 2004
PubMed
Summary

Buffer protonation significantly impacts pharmacologic ligand-macromolecule affinity measurements. This study highlights the need to consider buffer ionization enthalpy (Delta H(buffer)) for accurate binding constants like K(d) or K(i).

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Physical Chemistry

Background:

  • Ligand-macromolecule interactions are fundamental in pharmacology and molecular biology.
  • Affinity constants (K(d), K(i)) are standard metrics, often neglecting buffer effects.
  • Buffer ionization can influence measured binding thermodynamics.

Purpose of the Study:

  • To investigate the impact of buffer protonation on ligand-macromolecule binding affinity.
  • To demonstrate the necessity of accounting for buffer ionization enthalpy (Delta H(buffer)) in affinity measurements.
  • To provide a more accurate characterization of binding interactions.

Main Methods:

  • Utilized isothermal titration microcalorimetry (ITC) for precise thermodynamic measurements.
  • Employed a model system: competitive inhibition of cytidine 2'-monophosphate (2'-CMP) binding to RNase-A.

Related Experiment Videos

  • Conducted measurements across buffers with varying ionization properties (Delta H(buffer)).

    • Main Results:

    • Observed significant variations in measured affinity constants based on buffer protonation.
    • Demonstrated a direct correlation between buffer ionization enthalpy and binding affinity.
    • Quantified the protonation effect on the thermodynamics of 2'-CMP binding to RNase-A.

    Conclusions:

    • Buffer protonation is a critical, often overlooked, factor in determining ligand-macromolecule binding affinity.
    • Accurate affinity measurements require consideration of the buffer's thermodynamic properties, specifically Delta H(buffer).
    • This research advocates for incorporating buffer effects into standard protocols for characterizing molecular interactions.