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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.


Three possible cuprous complexes can form during the BCA protein assay: a) BCA-Cu1+-BCA; b) BCA-Cu1+-NTPB; c) NTPBCu1+-NTPB.
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Figure 1: Three possible cuprous complexes can form during the BCA protein assay: a) BCA-Cu1+-BCA; b) BCA-Cu1+-NTPB; c) NTPBCu1+-NTPB.

Mentions: Since the original work of Smith et al. (1985) [1], bicinchoninic acid (BCA) has been widely used to determine protein concentration because of its ease of use, high sensitivity and tolerance of interfering species [2, 3]. This assay is based on two chemical reactions. The first is the reduction of cupric ions (Cu2+) to cuprous ions (Cu1+) by the peptide bonds, known as the biuret reaction, and by some specific residues in an alkaline environment [1]. The second step is the chelation of one Cu1+ with two BCA molecules (Fig. 1a), forming an intense purple complex, which has a peak absorbance at 562nm. The protein concentration in a solution is determined by comparing this absorbance with a standard curve of absorbance from varying bovine serum albumin (BSA) [1]. For the Micro BCATM Assay Kit of Pierce (23235#), the standard curve appears nonlinear above the working range of 0.5 to 20 µg/mL protein. The reason for this nonlinearity has not been investigated. When we increased the concentration of BSA up to 6000 µg/mL, an unexpected absorbance maximum was observed. The molar concentrations of cupric ions, BCA molecules and peptide bonds were calculated from the given contents of the kit. It was found that the absorbance maximum appeared where the peptide bonds were about six times more concentrated than the cupric ions. It is reasonable to assume that the excess peptide bonds compete with the BCA for binding to cuprous ion, thereby reducing production of the chromogenic group. We describe a study of this competition by experiments and theoretical modeling.


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)

Three possible cuprous complexes can form during the BCA protein assay: a) BCA-Cu1+-BCA; b) BCA-Cu1+-NTPB; c) NTPBCu1+-NTPB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Three possible cuprous complexes can form during the BCA protein assay: a) BCA-Cu1+-BCA; b) BCA-Cu1+-NTPB; c) NTPBCu1+-NTPB.
Mentions: Since the original work of Smith et al. (1985) [1], bicinchoninic acid (BCA) has been widely used to determine protein concentration because of its ease of use, high sensitivity and tolerance of interfering species [2, 3]. This assay is based on two chemical reactions. The first is the reduction of cupric ions (Cu2+) to cuprous ions (Cu1+) by the peptide bonds, known as the biuret reaction, and by some specific residues in an alkaline environment [1]. The second step is the chelation of one Cu1+ with two BCA molecules (Fig. 1a), forming an intense purple complex, which has a peak absorbance at 562nm. The protein concentration in a solution is determined by comparing this absorbance with a standard curve of absorbance from varying bovine serum albumin (BSA) [1]. For the Micro BCATM Assay Kit of Pierce (23235#), the standard curve appears nonlinear above the working range of 0.5 to 20 µg/mL protein. The reason for this nonlinearity has not been investigated. When we increased the concentration of BSA up to 6000 µg/mL, an unexpected absorbance maximum was observed. The molar concentrations of cupric ions, BCA molecules and peptide bonds were calculated from the given contents of the kit. It was found that the absorbance maximum appeared where the peptide bonds were about six times more concentrated than the cupric ions. It is reasonable to assume that the excess peptide bonds compete with the BCA for binding to cuprous ion, thereby reducing production of the chromogenic group. We describe a study of this competition by experiments and theoretical modeling.

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.