Two-peak approximation in kinetic capillary electrophoresis

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Kinetic capillary electrophoresis (KCE) is a homogeneous kinetic affinity method for measuring rate constants of formation (k(+)) and dissociation (k(-)) of non-covalent biomolecular complexes.
• Current parameter-based approaches rely on approximate solutions to mass transfer equations, which can be challenging to derive.

Purpose of the Study:

• To introduce a novel approximate analytical solution for mass transfer equations in KCE, termed the "two-peak approximation."
• To develop a corresponding parameter-based method for determining k(+) and k(-) using this approximation.
• To validate the accuracy and applicability of the two-peak approximation.

Main Methods:

• Developed an approximate analytical solution for mass transfer equations in KCE (the "two-peak approximation").
• Applied this approximation to KCE methods where two peaks are identifiable and the concentration of one binding partner remains constant.
• Validated the approximation using simulated propagation patterns derived from an exact solution of mass transfer equations.

Main Results:

• The two-peak approximation was found to be accurate for determining k(+) and k(-).
• The method achieves a relative error of less than 10% when two peaks are identifiable in the KCE electropherogram.
• The approximation is applicable to the macroscopic approach to studying kinetics at equilibrium (MASKE), including fast, slow, and intermediate-rate interactions.

Conclusions:

• The two-peak approximation offers a simplified and accurate method for extracting kinetic rate constants from KCE data.
• This method enhances the utility of MASKE, making it a practical tool for studying a wide range of non-covalent interactions without complex fitting procedures.
• The findings complete a set of fitting-free methods for processing MASKE data, simplifying kinetic analysis.