Abstract
The protein binding of Naphthol Yellow S to bovine serum albumin (BSA) was examined and three pairs of parameters (binding numbers and constants) were obtained by equilibrium dialysis and Scatchard plot. These were n=1, 2, and 8, and ∞ (experimentally), 2.4×105M-1, and 1×103M-1, respectively. Though the spectra of Naphthol Yellow S showed change in absorbance by addition of BSA and the spectra with or without BSA intersected each other at a certain wavelength, not showing an isosbestic point. The results from equilibrium dialysis showed that two classes of binding were contributing greatly to the spectral change. An attempt was made to estimate the molar absorptivities of these two species. The calculated absorbances from the molar absorptivities of free or bound dye and their concentrations agreed well with those of observed. Thus the inference validity was ascertained. When the protein bindings are studied spectrophotometrically, the molar absorptivities should also be taken into consideration as so many classes if they have two or more binding classes. In the system of Naphthol Yellow S-BSA, the third binding was weak and masked by the first and second bindings, and the molar absorptivity of the third binding was not obtained as yet.
