Correction for light absorption in fluorescence studies of protein-ligand interactions.
It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.
D008024:Ligands / Q000032:analysis*; D008027:Light; D008962:Models, Theoretical; D011506:Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D011506:Proteins