- The Biuret Assay, also known as Biuret Method, is a classical biochemical technique used for the quantification of proteins based on the presence of peptide bonds.
- It is named after the Biuret reaction, a chemical reaction in which copper(II) ions form a violet-colored complex with compounds containing two or more peptide bonds under alkaline conditions. This colorimetric change can be measured spectrophotometrically, providing a direct correlation between absorbance and protein concentration.
- In the Biuret assay, a reagent containing copper sulfate is added to the protein sample in an alkaline medium, typically maintained by sodium hydroxide. To stabilize the copper ions and prevent their precipitation, chelating agents such as sodium potassium tartrate are included. When proteins are present, the copper ions coordinate with the nitrogen atoms of the peptide backbone, producing a characteristic violet or purple color. The intensity of this color is measured at wavelengths between 540 and 560 nm, and it increases proportionally with the concentration of protein in the solution.
- This method is relatively simple, robust, and highly specific to proteins, as it does not respond to free amino acids or small peptides lacking sufficient peptide bonds. However, its sensitivity is limited compared to modern assays such as the Bradford, Lowry, or BCA methods. The Biuret assay typically requires protein concentrations above 1 mg/mL, making it less suitable for samples where protein is present in low amounts. Moreover, substances that interfere with the alkaline environment or form complexes with copper ions—such as ammonia or strong chelators—can affect assay accuracy.
- Despite these limitations, the Biuret Assay remains useful in certain contexts, especially in clinical biochemistry laboratories where it is routinely used to measure total protein levels in serum and plasma samples. Its high reproducibility and tolerance to various sample matrices make it reliable for large-scale or automated workflows. While no longer the most commonly used assay in molecular biology research, the Biuret Assay continues to serve as a foundational technique in protein chemistry.
