Determination of Protein by the Biuret Reaction

The Biuret reaction is a colorimetric method used for the quantitative determination of proteins. It is based on the reaction of peptide bonds in proteins with copper(II) ions in an alkaline medium, producing a violet-colored coordination complex. The intensity of the violet color is related to the amount of protein present in the sample.

Principle

Proteins contain numerous peptide bonds (–CO–NH–). In an alkaline medium, peptide bonds interact with Cu²⁺ ions to form a violet-colored coordination complex. Under suitable experimental conditions, the intensity of the color increases with increasing protein concentration.

The absorbance of the colored complex is measured using a spectrophotometer at approximately 540 nm. A standard curve prepared using known concentrations of a standard protein is then used to determine the protein concentration of the unknown sample.

Requirements

• Protein sample solution
• Biuret reagent containing copper(II) sulfate, sodium hydroxide, and potassium sodium tartrate
• Standard protein solution, such as bovine serum albumin (BSA)
• Distilled or deionized water
• Test tubes or suitable reaction tubes
• Pipettes and measuring equipment
• Spectrophotometer capable of measuring absorbance at about 540 nm

Procedure

1. Prepare a series of standard protein solutions of known concentrations.
2. Prepare a reagent blank containing the Biuret reagent and solvent but no protein.
3. Add an appropriate volume of Biuret reagent to each protein standard, the unknown sample, and the reagent blank.
4. Mix the solutions thoroughly and allow the reaction to develop for the recommended time, commonly about 20–30 minutes at room temperature.
5. Measure the absorbance of the standards and unknown sample at approximately 540 nm, using the reagent blank to establish the spectrophotometer baseline.
6. Plot a standard curve of absorbance versus protein concentration.
7. Determine the protein concentration of the unknown sample from the standard curve. If the sample was diluted before analysis, apply the appropriate dilution factor.

Role of Components of Biuret Reagent

• Copper(II) sulfate: Provides Cu²⁺ ions that participate in the formation of the violet protein–copper complex.
• Sodium hydroxide: Provides the alkaline medium required for the reaction.
• Potassium sodium tartrate: Helps keep copper ions in solution and reduces the tendency of copper hydroxide to precipitate under alkaline conditions.

Applications

• Quantitative estimation of total protein in biological samples.
• Determination of total protein concentration in serum and other laboratory samples.
• Protein analysis in food and biochemical research.
• Routine estimation of protein concentration in laboratory studies.

Advantages

• Simple and relatively inexpensive method.
• Suitable for quantitative estimation of protein.
• Less affected by some interfering substances than certain other colorimetric protein assays.
• Provides a reasonably reproducible response when experimental conditions are properly controlled.

Limitations

• The Biuret method is less sensitive than more sensitive protein assays such as the Lowry and Bradford methods.
• It generally requires a relatively higher concentration of protein for reliable measurement.
• Substances capable of binding copper ions or strongly affecting the alkaline reaction may interfere with the assay.

Key Point: The Biuret method is based on the formation of a violet-colored complex between peptide bonds and Cu²⁺ ions in an alkaline medium. The absorbance of the complex is measured at approximately 540 nm and is compared with a standard protein calibration curve to determine the protein concentration of an unknown sample.