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Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

Huang T, Long M, Huo B - Open Biomed Eng J (2010)

Bottom Line: 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.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-Cu(1+)-NTPB that might exist in the final solution.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomechanics and Bioengineering and Key Laboratory of Microgravity (National Microgravity Laboratory), Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, People's Republic of China.

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-Cu(1+)-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-Cu(1+)-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.

No MeSH data available.


Parameter b fitted by Eq. (8) for the corresponding ρB0 is denoted by the points. For constant initial concentration of Cu2+ (0.4mM), the variation of b with initial concentration of BCA was calculated using Eq. (9) (parameters b0, b1, c come from Table 2). The determined coefficient R2 of each fitting was greater than 0.97
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Figure 4: Parameter b fitted by Eq. (8) for the corresponding ρB0 is denoted by the points. For constant initial concentration of Cu2+ (0.4mM), the variation of b with initial concentration of BCA was calculated using Eq. (9) (parameters b0, b1, c come from Table 2). The determined coefficient R2 of each fitting was greater than 0.97

Mentions: The parameter, b can be considered as an integrated equilibrium constant, which reflects the effect of some factors of amino acid composition and the initial concentrations of BCA and Cu2+ on the reductive reaction. For each set of experimental data with the same initial concentration of BCA, the value of b was determined by fitting it with Eq. (8), in which the dimensionless ratio ρC1t/ρC20 could be obtained from the fitting result with Eq. (5) and Eq. (7). The value of b as a function of the initial concentration of BCA is plotted in Fig. (4). As expected, b increased with the initial concentration of BCA to a final constant value. We use another empirical exponential function to describe the effect of BCA concentration on b:


Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

Huang T, Long M, Huo B - Open Biomed Eng J (2010)

Parameter b fitted by Eq. (8) for the corresponding ρB0 is denoted by the points. For constant initial concentration of Cu2+ (0.4mM), the variation of b with initial concentration of BCA was calculated using Eq. (9) (parameters b0, b1, c come from Table 2). The determined coefficient R2 of each fitting was greater than 0.97
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102443&req=5

Figure 4: Parameter b fitted by Eq. (8) for the corresponding ρB0 is denoted by the points. For constant initial concentration of Cu2+ (0.4mM), the variation of b with initial concentration of BCA was calculated using Eq. (9) (parameters b0, b1, c come from Table 2). The determined coefficient R2 of each fitting was greater than 0.97
Mentions: The parameter, b can be considered as an integrated equilibrium constant, which reflects the effect of some factors of amino acid composition and the initial concentrations of BCA and Cu2+ on the reductive reaction. For each set of experimental data with the same initial concentration of BCA, the value of b was determined by fitting it with Eq. (8), in which the dimensionless ratio ρC1t/ρC20 could be obtained from the fitting result with Eq. (5) and Eq. (7). The value of b as a function of the initial concentration of BCA is plotted in Fig. (4). As expected, b increased with the initial concentration of BCA to a final constant value. We use another empirical exponential function to describe the effect of BCA concentration on b:

Bottom Line: 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.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-Cu(1+)-NTPB that might exist in the final solution.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomechanics and Bioengineering and Key Laboratory of Microgravity (National Microgravity Laboratory), Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, People's Republic of China.

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-Cu(1+)-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-Cu(1+)-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.

No MeSH data available.