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A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Published on: October 16, 2015
Analytic models for nonlinear curve-fitting of forward-rate binding data, with applications to hemoglobin
1Division of Computer Research and Technology, National Institutes of Health, Bethesda, MD 20892.
Journal of Biochemical and Biophysical Methods
|October 1, 1992
Summary
This study presents a new model for stepwise ligand binding in hemoglobin, enabling analysis of intermediate states during kinetic reactions. The method determines forward rate ratios and can incorporate time data for comprehensive kinetic analysis.
Area of Science:
- Biochemistry
- Chemical Kinetics
- Molecular Biophysics
Background:
- Hemoglobin exhibits complex intermediate ligation states during ligand binding.
- Understanding these states is crucial for elucidating hemoglobin function and kinetics.
- Previous methods had limitations in fully characterizing these intermediate states.
Purpose of the Study:
- To develop and validate a model for stepwise ligand binding to hemoglobin.
- To analyze intermediate ligation states under equilibrium and kinetic conditions.
- To determine forward rate ratios and scale factors for ligand binding kinetics.
Main Methods:
- A stepwise ligand binding model was derived, assuming negligible reverse reactions.
- The model was fitted to normalized concentration versus saturation data.
- An auxiliary relation was optionally used with time information to determine rate scale factors.
Main Results:
- The procedure successfully determined forward rate ratios for hemoglobin.
- The model was applied to carbon monoxide binding kinetics.
- The methodology is adaptable for other ligands, such as ferricyanide.
Conclusions:
- The developed model provides a robust method for analyzing hemoglobin ligation states.
- This approach enhances the understanding of hemoglobin's kinetic reaction mechanisms.
- The procedure offers a valuable tool for studying protein-ligand interactions.
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