A linear regression method for the study of the Coomassie brilliant blue protein assay

• Published in: Talanta (Oxford) 1997-05, Vol.44 (5), p.923-930
• Main Authors: Wei, Yong-ju, Li, Ke-an, Tong, Shen-yang
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Coomassie brilliant blue protein assay; Fundamental and applied biological sciences. Psychology; Solution equilibria; Spectrophotometry
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/S0039-9140(96)02140-6

The interactions of Coomassie brilliant blue G-250 (CBB) with bovine serum albumin (BSA) and γ-globulin at low pH are investigated by a spectrophotometric method. It is considered that the binding of CBB to protein is because of the weak interactions (ionic, van der Waals, hydrogen bonding, and hydrophobic). The solution equilibria involving the binding of three dye species (blue, green, and red) to protein are treated in the same way as Ringbom model used in the treatment of complexation in analytical chemistry. Based on this treatment, the formation of an isosbestic point in the absorption spectra of CBB-BSA mixtures is discussed, two mathematical models for the description of the CBB protein assay are developed. The first model is a nonlinear equation which is rigorous in theory but unreliable in use because of its optimization procedure. The second model based on an approximation is a linear equation, it allows to estimate apparent binding constant, maximum binding number, and molar absorptivity of bound dye from assay data by a linear regression method. The results of the linear regression operations are reasonable and in agreement with experimental findings. Factors which influence the sensitivity of the CBB protein assay are studied using this method. Ionic strength and acidity are found to have significant effect on the binding of CBB to protein.