In the Biuret assay for proteins, how many peptide bonds are required for a positive reaction?

• A. One peptide bond
• B. Two peptide bonds
• C. Three peptide bonds
• D. Four peptide bonds

Answer: (B)

The signal in the Biuret test comes from copper ions forming a complex with peptide bonds in an alkaline solution. This complex requires at least two peptide bonds to coordinate properly, so molecules with two or more bonds give the characteristic color change. A single peptide bond—found in a dipeptide—does not provide enough binding sites, so it remains negative. Therefore, the minimum number of peptide bonds needed for a positive reaction is two, which is why the test becomes positive for tripeptides, proteins, and any larger peptide chains.