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Extracting equilibrium constants from kinetically limited reacting systems.
John J Correia1, Walter F Stafford
1Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA.
Methods in Enzymology
|March 18, 2009
Summary
This study demonstrates direct boundary fitting for monomer-dimer systems with slow kinetics using sedimentation velocity data. The kinetic integrator in Sedanal allows for precise determination of dissociation rate constants (k(off)) within specific relaxation time ranges.
Area of Science:
- Biophysical Chemistry
- Biochemistry
- Analytical Chemistry
Background:
- Slow kinetics in reversible reactions distort reaction boundary shapes.
- Previous studies indicated rapid reaction behavior in monomer-dimer systems persists until relaxation times exceed 100 seconds.
Purpose of the Study:
- To present a tutorial on direct boundary fitting of sedimentation velocity data for monomer-dimer systems exhibiting kinetic effects.
- To utilize a kinetic integrator to determine dissociation rate constants (k(off)) and their uncertainties.
Main Methods:
- Sedimentation velocity analysis.
- Direct boundary fitting using a kinetic integrator feature in Sedanal software.
- Analysis of monomer-dimer systems with varying relaxation times.
Main Results:
- The kinetic integrator allows fitting for k(off) values with 95% confidence intervals.
- Well-determined k(off) values are achievable for relaxation times between approximately 70 and 33,000 seconds.
- For shorter relaxation times, only the equilibrium constant (K) is uniquely determined; for longer times, k(off) cannot be uniquely determined.
Conclusions:
- Direct boundary fitting with kinetic integration provides a robust method for studying slow kinetics in monomer-dimer systems.
- The method enables accurate determination of dissociation rate constants within a defined range of relaxation times.
- Understanding kinetic limitations is crucial for accurate interpretation of sedimentation velocity data.
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