Estimation of Protein by the Biuret Method

Principle
This is the most commonly used method based on the fact that the - CO - NH (peptide) group of proteins form a purple complex with copper ions in an alkaline medium. Since all proteins contain the peptide bond, the method is fairly specific and there is little interference from other compounds. Some substances like urea and biuret interfere because they possess the - CO – NH - group. Other interfering materials are reducing sugarlike glucose, which interacts with Cu⁺³ ions (cupric) in the reagent.

Reagents Required

1. Biuret reagent: Dissolve
   1.5 gm of CuSO₄ and 4.5 gms of Na-K tartrate in 250 mL 0.2 N NaOH solution. Add 2.5 gms of KI and make up the volume to 500 mL with 0.2 N NaOH.
2. 0.2 N NaOH.
3. Protein standard solution:
   Dissolve 500 mg of egg albumin in 50 mL of H₂O. Make up the volume to 100 mL to get the final concentration of 5 mg/mL.

Procedure
Pipette out standard protein solution into a series of tubes — 0.0, 0.2, ..., 1 mL and make up the total volume to 4 mL by adding water. The blank tube will have only 4 mL of water. Add 6 mL of biuret reagent to each tube and mix well. Keep the tubes at 37°C for 10 minutes during which a purple color will develop. The optical density of each tube is measured at 52.0 nm (green filter) using the blank reagent. Draw the graph to the known concentrate of a protein in an unknown solution

Result:
The given sample contains .......... mg of protein.