Bromocresol green

Bromocresol green is a pH-sensitive triphenylmethane dye commonly used for the determination of protein and albumin in serum. Bromocresol green is a bio-based dye with a yellow-green to blue-green color. Bromocresol green turns yellow (λmax=435 nm, protonated form) when placed in acidic solution (e.g. pH=4.15), and turns blue in basic solution (λmax=615 nm, deprotonated form). Bromocresol green is widely used as a pH indicator in the field of biochemical analysis. In addition, Bromocresol green is also used to detect the concentration of molecules such as creatinine, and to judge the viability of cells^[1][2][3][4]. In Vitro:Determination of Bromocresol Green Binding with Human Serum Albumin^[1]
(1) Reagent preparation: Prepare a 0.05 M phosphate buffer or any other needed buffer, adding NaCl if necessary to maintain an ionic strength of at least 0.03. Dissolve Bromocresol Green in water and adjust the pH with NaOH to ensure it forms a divalent anionic form, creating a 0.04% stock solution.
(2) Sample preparation: Prepare a solution of human serum albumin. After determining the nitrogen content using the Kjeldahl method, dilute it with the required buffer to create a solution with an appropriate concentration of albumin. Depending on the experimental design, prepare buffers of different pH values and different concentrations of Bromocresol Green solution.
(3) Binding experiment: Under set pH conditions (e.g., pH 6.95), add a certain amount of Bromocresol Green to a solution containing a fixed concentration of human serum albumin. Measure the changes in absorbance of the solution at specific wavelengths using a spectrophotometer, and record the data. Pay special attention to changes at 615 nm, as this is where the maximum absorption difference between the free form and the albumin-bound state of Bromocresol Green occurs. Separate the free and bound indicators using ultrafiltration and measure the absorbance again.
(4) Data analysis: Analyze the absorbance data to determine the binding constant and the number of binding sites. Use the Scatchard plot analysis method to estimate the number of binding sites and their respective binding constants. Compare the experimental data with theoretical curves to evaluate the effect of different pH values on binding.