SPECTRAL STUDY OF INTERACTION OF THYMOL BLUE WITH PROTEIN IN ACIDIC SOLUTION

The interactions of thymol blue (TB) with bovine serum albumin (BSA) a nd gamma-globulin (gamma-G) in acidic solution were studied by a spect rophotometric method. The absorption spectra of TB at about pH 1.4 app ears as two absorption peaks at 430 and 545 nm. Addition of protein ca uses an increase in absorbance at 440 nm and a decrease at 545 nm. An isosbestic point is formed at 495 nm. This spectral feature was explai ned based on the consideration of the dye-binding mechanism and soluti on equilibria. It was found that there is no linear relationship betwe en Delta A (absorbance change) and c(p) (protein concentration). Inste ad, an excellent linearity between Delta A(-1) and c(p)(-1) was observ ed. A linear regression equation was derived to interpret this finding and, from this equation the apparent binding constant and molar absor ptivity of the dye-protein complex were calculated. The Sandell index was used to express the sensitivity of a dye-binding protein assay, an d the Sandell indices for BSA and gamma-G were determined to be 0.18 a nd 0.38 mu g cm(-2), respectively. This sensitivity is higher than tha t of the bromocresol green method, but lower than that of the Coomassi e brilliant blue assay.