Protein Determination Reagents

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Various Methods of Protein Determination

The bicinchoninic acid (BCA) assay, Bradford assay, and Lowry assay are common methods for determining the total protein content of samples extracted from cells and tissues. Four assay methods, including the pyrogallol red-molybdenum assay, are described below.

Bicinchoninic Acid (BCA) Assay

The BCA assay is an improved version of the Lowry assay described below. This method allows quantitative protein analysis even when detergents commonly used to solubilize proteins, such as sodium dodecyl sulfate (SDS) and Triton-X, are present in the sample¹⁾. The detection range of the BCA assay is 1 to 2,000 ng/mL.

Principle of the BCA Assay

Proteins form a chelate complex with Cu(II) under alkaline conditions, and Cu(II) is reduced to Cu(I) by cysteine, tyrosine, and tryptophan in the protein. The amount of Cu(I) produced by the reduction is proportional to the amount of protein.

When BCA is added to Cu(I), two molecules of BCA chelate Cu(I), forming a blue-violet complex with strong absorption at 562 nm. The absorbance at this wavelength is measured, and colorimetric protein determination is performed using a standard curve prepared with a standard protein.

The Biuret and Lowry assays are based on the same principle.

Advantages of the BCA Assay

The detection range of the BCA assay is 1 to 2,000 ng/mL, showing linearity over a wide concentration range and high detection sensitivity. In addition, the color reaction with BCA is relatively insusceptible to detergents or protein denaturants such as urea and guanidine hydrochloride.

Disadvantages of the BCA Assay

Since the BCA assay involves the reduction of Cu(II) to Cu(I) and the chelate formation between BCA and Cu(I), chelating reagents such as EDTA and reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) interfere with the quantitative analysis of proteins.

Bradford Assay

The Bradford assay uses the triphenylmethane dye Coomassie Brilliant Blue G-250 (CBB G-250). The detection range of the Bradford assay is 10 to 2,000 ng/mL.

Principle of the Bradford Assay

When added to a protein solution under acidic conditions, CBB G-250 non-covalently binds to proteins through electrostatic interactions with basic amino acid residues (arginine, lysine, histidine) and N-terminal amino acids and through hydrophobic interactions with aromatic amino acids.

In this process, the maximum absorption wavelength of CBB G-250 shifts from 465 nm to 595 nm, and the color tone changes from reddish purple to blue. Proteins can be quantified by measuring the change in absorbance at 595 nm.

Advantages of the Bradford Assay

The procedure is very simple, as the assay can be performed by mixing the protein solutions to be assayed with CBB G-250 and allowing them to stand at room temperature. Furthermore, reducing agents or chelating agents in the sample do not significantly interfere with the color reaction of CBB G-250. This assay is useful when the BCA assay cannot be used due to a reducing agent or chelating agent in the sample.

Disadvantages of the Bradford Assay

Detergents in the sample interfere with the assay. When this is the case, the sample should be diluted to reduce the concentration of detergent, or a different assay based on a different principle should be used.

Lowry assay

The Biuret reagent and Folin-Ciocalteu reagent (phosphomolybdic acid and phosphotungstic acid dissolved in acidic solution) are used in the Lowry assay. The detection range of the Lowry assay is approximately 1 to 1,500 ng/mL.

Principle of the Lowry assay

When the Biuret reagent is added to a protein solution under alkaline conditions, Cu(II) in the Biuret reagent forms a chelate complex with the peptide bonds that make up the protein.

Subsequently, the Folin-Ciocalteu reagent is added. The phosphotungstic acid and phosphomolybdic acid are reduced by tryptophan, tyrosine, and cysteine in the protein, and the protein solution turns blue. The absorbance at 750 nm is measured, and protein determination is performed using a standard curve prepared with a standard protein.

Advantages of the Lowry assay

Compared to the Biuret assay, the Lowry assay has a higher detection sensitivity and broader applicability.

Disadvantages of the Lowry assay

The reaction between proteins and the Biuret and Folin-Ciocalteu reagents takes 30-60 minutes, which is longer than other assay methods. In addition, since the Lowry assay involves a reduction reaction, reducing agents (thiols, phenols, etc.) interfere with the color reaction. The Lowry assay is incompatible with components of the buffer solution often used to prepare protein samples (detergents, glycerol, Tricine, chelating agent, and Tris), and precipitates may form.
The BCA assay is an improved assay method based on the Lowry assay.

Pyrogallol Red-Molybdenum Assay

This assay is used to quantify total protein in urine and cerebrospinal fluid samples. Pyrogallol red (PR) and the metal ion molybdenum (VI) form a red chelate complex with a maximum absorption wavelength at 470 nm. Under acidic conditions, the chelate complex binds to the protein, resulting in a shift of the maximum absorption wavelength to approximately 600 nm²⁾. The amount of protein in the sample is determined by measuring the absorbance at 600 nm.

References

1. Suzuki, Y.: Bunseki, 1, 2(2018).
   Analysis of Proteins, Nucleic Acids, and Genes: Quantitative Analytical Methods of Total Proteins (Japanese)
2. Watanabe, N. et al.: J. Med. Tech., 30(7), 778(1986).
   Protein determination in urine and cerebrospinal fluid using pyrogallol red-molybdenum complex reagent (Japanese)