RESEARCH ARTICLE
Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay
Tao Huang, Mian Long, Bo Huo*
Article Information
Identifiers and Pagination:
Year: 2010Volume: 4
First Page: 271
Last Page: 278
Publisher ID: TOBEJ-4-271
DOI: 10.2174/1874120701004010271
Article History:
Received Date: 26/6/2010Revision Received Date: 11/8/2010
Acceptance Date: 19/8/2010
Electronic publication date: 3/11/2010
Collection year: 2010
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution, and reproduction in any medium, provided the work is properly cited.
Abstract
Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.
