ABSTRACT
I. INTRODUCTION Many techniques have been developed to measure the affinity of one molecule for another, to determine apparent equilibrium constants for molecular association. All experimental techniques used for measuring equilibrium constants base on titrating changes in physicochemical properties of molecules depending on the concentration of the substrate. The system response has to be different for free versus complexed molecules. Changes in size, charge, and other properties might result in measurable differences in diffusion rate (immunodiffusion, equilibrium dialysis), molecular weight (size exclusion methods), sedimentation (ultracentrifugation), solubility, spectroscopic properties (fluorescence quenching, spectral shift), and electrophoretic migration. They can be measured using spectroscopic methods, calorimetry, potentiometry, nuclear magnetic resonance (1), ultrafiltration, or phase solubility experiments (2). Conventional methods involve the separation of bound and free molecules through the use of filtration or equilibrium dialysis and subsequent detection by spectroscopic methods as well as fluorescence measurement or scintillation counting. The components of interest are radioactively or fluorescently labeled (3).
