Reaction between pH indicators with a large pK_a value and human serum albumin and its application to the determination of the serum albumin concentration

Abstract

The reaction between four pH indicators with a large pK_a value and human serum albumin and its application to the determination of the serum albumin concentration were studied. In the reaction of Bromothymol Blue (BTB), Phenol Red, Cresol Red and Thymol Blue with serum albumin, although a change in the color shade did not occur, the absorbance of the reaction mixture decreased. Since this absorbance decrease was largest in the reaction of BTB(the molar absorbance decrease at pH 7.4 was 7.59×10⁴ l mol⁻¹ cm⁻¹), which was sufficiently sensitive to measure the serum albumin concentration, the BTB color reagent at pH 7.4 was applied to its determination. The measurement values by the BTB method correlated with those by the dye-binding method using the Bromocresol Green method(r = 0.965, y = 1.21x−4.25, n = 90) and with those by a method using Bromocresol Purple(r = 0.953, y = 1.04x + 3.56, n = 90). The mean value of the BTB method (41.8 g/l) was higher than those of the Bromocresol Green method (38.1 g/l) and the Bromocresol Purple method(36.6 g/l). Since the reaction of BTB was carried out at pH 7.4, where serum protein does not precipitate, the addition of a detergent for preventing the precipitation of serum protein is not required. Therefore, the use of this color reagent contributes to decreasing water pollution by the detergent and acid used in the conventional methods.