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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.


Schematic drawing to display the chemical species in a solution during the BCA protein assay. Species enclosed by a circle are the reactants. The peptide bonds and specific residues reduce cupric ions to cuprous ions, which form two complexes with peptide bonds and BCA. Peptide bonds can also form a complex with cupric ions (biuret reaction).
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Figure 6: Schematic drawing to display the chemical species in a solution during the BCA protein assay. Species enclosed by a circle are the reactants. The peptide bonds and specific residues reduce cupric ions to cuprous ions, which form two complexes with peptide bonds and BCA. Peptide bonds can also form a complex with cupric ions (biuret reaction).

Mentions: Based on experimental results, we proposed a competition of peptide bonds with BCA molecules for binding to cuprous ions and established a theoretical model to describe quantitatively the interactions between the substances in the BCA protein assay (Fig. 6). In the competition of peptide bonds with BCA for binding to cuprous ions, both tetradentate complexes of BCA-Cu1+-NTPB and NTPB-Cu1+-NTPB were considered as possibilities, but the fitted data indicated that only the former complex existed. The steric repulsion in the latter complex of long peptide chains might be the main reason for the inexistence of NTPB-Cu1+-NTPB.


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)

Schematic drawing to display the chemical species in a solution during the BCA protein assay. Species enclosed by a circle are the reactants. The peptide bonds and specific residues reduce cupric ions to cuprous ions, which form two complexes with peptide bonds and BCA. Peptide bonds can also form a complex with cupric ions (biuret reaction).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Schematic drawing to display the chemical species in a solution during the BCA protein assay. Species enclosed by a circle are the reactants. The peptide bonds and specific residues reduce cupric ions to cuprous ions, which form two complexes with peptide bonds and BCA. Peptide bonds can also form a complex with cupric ions (biuret reaction).
Mentions: Based on experimental results, we proposed a competition of peptide bonds with BCA molecules for binding to cuprous ions and established a theoretical model to describe quantitatively the interactions between the substances in the BCA protein assay (Fig. 6). In the competition of peptide bonds with BCA for binding to cuprous ions, both tetradentate complexes of BCA-Cu1+-NTPB and NTPB-Cu1+-NTPB were considered as possibilities, but the fitted data indicated that only the former complex existed. The steric repulsion in the latter complex of long peptide chains might be the main reason for the inexistence of NTPB-Cu1+-NTPB.

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.