Methodological Comparison of Microsomal Protein Binding: Equilibrium Dialysis, Pre‑Saturation, and Flux Dialysis for Highly Bound Compounds
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June 02,2026
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Liver microsomes, containing key drug-metabolizing enzymes, are widely used in vitro for enzyme kinetic studies. Accurate estimation of kinetic parameters is often influenced by nonspecific binding of test compounds to microsomal proteins and membrane lipids, making determination of the free fraction (fu) critical for correcting binding-related biases. This study evaluated the performance of three methods—equilibrium dialysis, pre-saturation dialysis, and flux dialysis—across three microsomal protein concentrations (0.1, 0.5, and 1.0 mg/mL) using 13 highly bound compounds. The study also examined how equilibration time and protein stability during incubation affect fu results.
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