Ligand binding studies by high speed centrifugal precipitation and linear spectral summation using ultraviolet–visible absorption spectroscopy

In ligand-protein binding experiments the major challenge is to separate bound from free ligand. Equilibrium and gel filtration separation techniques are often hampered by competition for the ligand and non-specific binding. Biophysical assays have attempted to circumvent this problem using titration calorimetry and spectroscopic methods. However, insoluble ligands require solvents that can overwhelm the discernible enthalpic changes of the protein and ligands. Spectroscopic methods are effective but may suffer from insensitivity (NMR) or the need for a lipid analog e.g., fluorescence and electron paramagnetic resonance. Our purpose is to compare the standard fluorescence assay to a technique we call high speed centrifugal precipitation. High speed centrifugal precipitation is suited to ligands that are insoluble in aqueous. The method permits separation of insoluble free ligand from that bound to the protein. The concentration of the each fraction can be precisely measured by absorbance spectrophotometry. A second technique, linear spectral summation has been published for protein-ligand associations using fluorescence of labeled ligands [1]. Here, the method is altered for use with ultraviolet-visible (UV–Vis) absorption spectroscopy. If the ligand complex shows a shift in the peak absorption of >8 nm, the bound and free concentrations can be measured simultaneously. The composite spectra of the samples are fit by linearly scaling UV–Vis absorption spectra of pure bound and free components at each point. • Ligand- protein binding kinetics is accessible with an ordinary spectrophotometer. • Concentrations are accurately measured from molar extinction coefficients. • The methods are ideal for lipid ligands that show absorption spectral peaks shifts in the bound and free states and/or are insoluble.