科研之友
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摘要
Bicinchoninic acid (BCA) is widely used for determining the valence state of copper in biological systems and quantification of the total protein concentration (BCA assay). Despite its well-known high selectivity of Cu(I) over Cu(II), the exact formation constants for Cu(I)(BCA)(2)(3-) and Cu(II)(BCA)(2)(2-) complexes remain uncertain. These uncertainties, affect the correct interpretations of the roles of copper in biological processes and the BCA assay data. By studying the voltammetric behaviors of Cu(I)(BCA)(2)(3-) and Cu(II)(BCA)(2)(2-), we demonstrate that the apparent lack of redox reaction reversibility is caused by an adsorption wave of Cu(II)(BCA)(2)(2-). With the adsorption wave identified, we found that the Cu(I)/Cu(II) selectivity of BCA is essentially identical to another popular ligand, bathocuproinedisulfonic acid (BCS). Density functional theory calculation on the geometries of Cu(I)(BCA)(2)(3-) and Cu(II)(BCA)(2)(2-) rationalizes the preferential Cu(I) binding by BCA and the strong adsorption of the Cu(II)(BCA)(2)(2-) complex at the glassy carbon electrode. Based on the shift in the standard reduction potential of free Cu(II)/Cu(I) upon binding to BCA, we affirm that the formation constants for Cu(I)(BCA)(2)(3-) and Cu(II)(BCA)(2)(2-) are 10(17.2) and 10(8.9), respectively. Therefore, BCA can be chosen among various ligands for effective and reliable studies of the copper binding affinities of different biomolecules.
