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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.


Interpretation of the appearance of the nonlinearity and plateau of the absorbance with increasing initial protein concentration. Four dimensionless ratios were plotted and the calculation conditions were:                         ρB0=6.18 mM, ρC20=0.4 mM, b1=1.12, b0=0.596, c=0.00104, K1=0.198 mM−2, K2=0.0267 mM−2
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Figure 7: Interpretation of the appearance of the nonlinearity and plateau of the absorbance with increasing initial protein concentration. Four dimensionless ratios were plotted and the calculation conditions were: ρB0=6.18 mM, ρC20=0.4 mM, b1=1.12, b0=0.596, c=0.00104, K1=0.198 mM−2, K2=0.0267 mM−2

Mentions: The nonlinear relation between the protein concentration and the color production limits the measurement range of the BCA protein assay. The linear working range of the assay kit (23235#) is below 20 µg/mL. Although another assay kit from Pierce, (23225#) is claimed to be able to determine protein concentration in the range, 20 to 2000 µg/mL, its real maximum measurement limit is only about 100 µg/mL because in its procedure, the sample is diluted, one part with 20 parts of working reagent giving a 21-fold dilution. Therefore understanding the character of the nonlinearity will be helpful in extending the measurement of the BCA protein assay to higher protein concentrations. According to our results, the nonlinearity is caused by two factors, i.e. the reduction of Cu2+ and the competition of peptide bonds with BCA for binding to Cu1+. By giving a constant initial concentration of BCA, ρB0=6.18 mM, we plotted the curves of four dimensionless ratios, ρB2C1/ρC20,ρC1t/ρC20,ρB2C1/ρC1t and ρBC1P2/ρC1t, with respect to ρP0 in Fig. (7) and noted that ρB2C1ρC20=ρC1tρC20⋅ρB2C1ρC1t. It could be seen that for a low initial protein concentration, since ρB2C1/ρC1t was almost constant, it was the nonlinearity of the reduction reaction, but not the competition mechanism that dominated the nonlinearity of the color production. But at high protein concentrations, since ρC1t/ρC20 was almost equal to 1 and ρBC1P2/ρC1t increased dramatically, the reduction reaction was complete and the competition caused the nonlinearity of color formation.


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)

Interpretation of the appearance of the nonlinearity and plateau of the absorbance with increasing initial protein concentration. Four dimensionless ratios were plotted and the calculation conditions were:                         ρB0=6.18 mM, ρC20=0.4 mM, b1=1.12, b0=0.596, c=0.00104, K1=0.198 mM−2, K2=0.0267 mM−2
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Interpretation of the appearance of the nonlinearity and plateau of the absorbance with increasing initial protein concentration. Four dimensionless ratios were plotted and the calculation conditions were: ρB0=6.18 mM, ρC20=0.4 mM, b1=1.12, b0=0.596, c=0.00104, K1=0.198 mM−2, K2=0.0267 mM−2
Mentions: The nonlinear relation between the protein concentration and the color production limits the measurement range of the BCA protein assay. The linear working range of the assay kit (23235#) is below 20 µg/mL. Although another assay kit from Pierce, (23225#) is claimed to be able to determine protein concentration in the range, 20 to 2000 µg/mL, its real maximum measurement limit is only about 100 µg/mL because in its procedure, the sample is diluted, one part with 20 parts of working reagent giving a 21-fold dilution. Therefore understanding the character of the nonlinearity will be helpful in extending the measurement of the BCA protein assay to higher protein concentrations. According to our results, the nonlinearity is caused by two factors, i.e. the reduction of Cu2+ and the competition of peptide bonds with BCA for binding to Cu1+. By giving a constant initial concentration of BCA, ρB0=6.18 mM, we plotted the curves of four dimensionless ratios, ρB2C1/ρC20,ρC1t/ρC20,ρB2C1/ρC1t and ρBC1P2/ρC1t, with respect to ρP0 in Fig. (7) and noted that ρB2C1ρC20=ρC1tρC20⋅ρB2C1ρC1t. It could be seen that for a low initial protein concentration, since ρB2C1/ρC1t was almost constant, it was the nonlinearity of the reduction reaction, but not the competition mechanism that dominated the nonlinearity of the color production. But at high protein concentrations, since ρC1t/ρC20 was almost equal to 1 and ρBC1P2/ρC1t increased dramatically, the reduction reaction was complete and the competition caused the nonlinearity of color formation.

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.