Which reaction is used by most chemical methods for determining total protein?

• A. Molybdenum blue
• B. Ferri-Ferrocyanide
• C. Resorcinol-HCl
• D. Biuret

Answer: (D)

The main concept is that total protein is most reliably measured by detecting peptide bonds using the Biuret reaction. In alkaline solution, copper(II) ions form a violet complex with the nitrogen and carbonyl groups of peptide bonds. Since nearly all proteins contain peptide bonds, the amount of this copper–peptide complex is proportional to the total protein present. This makes the Biuret reaction the foundation for most chemical methods of protein quantification, and it also underpins many Lowry-type assays that incorporate the same copper-binding step. Other reagents correspond to alternative methods that rely on different chemistries (such as dye-binding or Folin–Ciocalteu-based steps) and are not the universal copper–peptide reaction used for total protein determination.