Accurate quantification of protein concentration is a foundational step in nearly all areas of biochemistry and molecular biology. Before researchers can study a protein, they must first determine how much of it is present in their solution. The Bicinchoninic Acid (BCA) assay is one of the most widely adopted and sensitive colorimetric methods used for this purpose, providing a reliable measure of the total protein content within a sample. Its popularity stems from its broad working range and the stability of the final colored product.
The Underlying Chemical Reaction
The mechanism of the BCA assay is a two-step chemical process that ultimately results in a visible color change, the intensity of which is proportional to the protein amount. This sequence begins with a reaction known as the biuret reaction, a well-established method for detecting the presence of peptide bonds. The peptide bonds found in protein chains act as reducing agents in the highly alkaline environment of the BCA reagent solution.
The first step involves the reduction of cupric ions (Cu\(^{2+}\)), which are initially present in the reagent as copper(II) sulfate. Protein chains chelate with these cupric ions, causing them to be reduced to cuprous ions (Cu\(^{+}\)) in a concentration-dependent manner. The amount of Cu\(^{+}\) generated is directly proportional to the number of peptide bonds present, meaning a higher protein concentration yields more Cu\(^{+}\). This initial reaction is also facilitated by the amino acid side chains of cysteine, cystine, tyrosine, and tryptophan residues.
The second and more sensitive step occurs when the newly formed cuprous ions react with the bicinchoninic acid (BCA) reagent. Two molecules of BCA chelate with a single cuprous ion (Cu\(^{+}\)) to form a stable, water-soluble complex. This chelation reaction produces a vivid purple color in the solution. Since the color intensity is directly proportional to the amount of Cu\(^{+}\) reduced, the final color serves as an accurate proxy for the protein concentration.
Quantifying Protein Concentration
The purple color generated by the BCA-copper complex must be translated into a numerical value to determine the protein concentration of the unknown sample. This measurement is achieved using a laboratory instrument called a spectrophotometer. The BCA-Cu\(^{+}\) complex exhibits a strong absorption of light at a wavelength of 562 nanometers (nm).
The spectrophotometer shines a beam of light at this specific wavelength through the sample and measures how much of the light is absorbed. A darker purple solution, indicating a higher protein concentration, will absorb more light, resulting in a higher absorbance reading. This relationship between color intensity and protein quantity is linear over a wide working range, allowing for reliable quantification.
To convert the measured absorbance reading into an actual concentration value, researchers must first construct a standard curve. This curve is created by testing a series of solutions containing known concentrations of a reference protein, typically Bovine Serum Albumin (BSA). The absorbance readings for these standards are plotted against their respective concentrations, and a best-fit line is calculated.
The absorbance reading of the unknown protein sample is then plotted onto this standard curve. By using the mathematical equation derived from the best-fit line, the concentration of the unknown sample can be accurately determined.
Common Sample Interferences
While the BCA assay is broadly compatible with many common laboratory detergents, its reliance on reduction chemistry makes it susceptible to interference from certain substances found in biological samples and buffers. Any compound that can also reduce cupric ions (Cu\(^{2+}\)) to cuprous ions (Cu\(^{+}\)) will artificially increase the measured color intensity. This leads to an overestimation of the protein concentration, as the instrument cannot distinguish between the Cu\(^{+}\) produced by the protein and the Cu\(^{+}\) produced by the contaminant.
A common class of interfering agents is reducing agents, such as Dithiothreitol (DTT) and \(\beta\)-mercaptoethanol (BME), which are often included in protein preparation buffers. Concentrations of DTT as low as 5 millimolar can disrupt the assay, causing a significant error in the final measurement. Conversely, chelating agents, which are designed to bind metal ions, interfere by sequestering the copper ions needed for the reaction, leading to an underestimation of the protein content.
Researchers employ several strategies to mitigate these interferences and ensure accurate results. The simplest approach is sample dilution, which reduces the concentration of the interfering substance to a level that no longer affects the assay. If the protein concentration is high enough, this dilution method is often sufficient.
Mitigation Strategies
For more challenging samples, the protein can be separated from the contaminants. Techniques like dialysis or precipitation using chemicals like trichloroacetic acid allow the protein to be re-dissolved in a compatible buffer for a clean measurement.