Measurements of Bovine Serum Albumin·Acid Violet 6B Binding by Dual-Wavelength Spectrophotometry

• Published in: YAKUGAKU ZASSHI Vol. 97; no. 1; pp. 36 - 40
• Main Authors: SEKIGUCHI, KEIJI, IWATSURU, MOTOHARU
• Format: Journal Article
• Language: Japanese
• Published: Japan The Pharmaceutical Society of Japan 01-01-1977
• Subjects: Acid Violet 6B; bovine serum albumin; Coloring Agents - analysis; double reciprocal plot; dual-wavelength spectrophotometry; metachromasy; Protein Binding; Scatchard's plot; Serum Albumin, Bovine - analysis; Spectrophotometry
• ISSN: 0031-6903; 1347-5231
• DOI: 10.1248/yakushi1947.97.1_36

The dual-wavelength spectrophotometer was employed to determine the protein-binding quantity between bovine serum albumin (BSA) and Acid Violet 6B (AV, C.I. 42640) which was used as a model compound. With the aid of a double-beam spectrophotometer, the two wavelengths (542 and 599 nm) were chosen on a basis of the magnitude of the absorbance change due to protein binding. The Beer's law held at 542 nm. Linearity was recognized between the AV concentrations and their absorbances, though the Beer's law did not hold at 599 nm. Therefore, the proportionality was observed between the AV concentrations and the absorbances measured with the dual-wavelength spectrophotometer by resetting the origin to the point of intersection of the two concentration-absorbance lines stated above. The double reciprocal plots were constructed from BSA concentrations and absorbances measured by the dual-wavelength mode, and the assumed absorbances of bound dye were read off by extrapolations. The amount of bound dye was estimated from the extrapolated values, and Scatchard's plots were made. From the linear part of the curves, the number of binding site(s) n and the binding constant K were determined to be 0.9-0.4 and 7.5×106 to 13×106M-1 respectively, depending on BSA concentration.