Estimation of Protein (Biuret Method) - Colorimetric Estimation

ESTIMATION OF PROTEIN (BIURET METHOD)

Aim

To estimate the amount of protein present in the given plasma sample.

Principle

The - CO - NH - group of protein forms a purple coloured complex with copper ion in alkaline medium. The colour intensity is measured at 540 nm. Since all proteins contain peptide bond, this method is fairly specific and there is little interference with other compounds.

Reagents required

Stock Biuret reagent

Dissolve 45g of Rochelle's salt (sodium potassium tartarate) in about 400ml of 0.2N sodium hydroxide and add 15g of copper sulphate. Stir it continuously until the solution is complete. Add 5g of potassium iodide and make upto 1 litre with 0.2N sodium hydroxide. Biuret solution for use

Dilute 200ml of stock biuret reagent to 1 litre with 0.2N sodium hydroxide containing 5 gm of potassium iodide.

Stock standard solution

1g of protein (egg albumin) is weighed and made upto 100ml with distilled water.

Concentration = l0mg / ml

Working standard solution

10ml of the stock is diluted to 100ml using distilled water.

Concentration = lmg / ml

Procedure

Estimation of protein

0.5 - 2.5 ml of standard protein solution is pipetted out into five different test tubes (S1- S5). The concentrations of protein in the tubes are 0.5 - 2.5 mg. 0.1 ml of plasma solution is taken in two test tubes labelled as T₁& T₂. The volume in all the tubes are made upto 5 ml using distilled water. A blank is also prepared simultaneously by adding 5 ml of distilled water. Then 3 ml of biuret reagent is added to all the test tubes including blank. The tubes are mixed well. The tubes are then maintained at room temperature for 10 minutes. The optical density is measured at 540nm.

From the values obtained, a standard graph is drawn using concentration of protein in X - axis and optical density in the Y - axis. From the graph the amount of protein present in the given plasma is calculated.

Protocol for protein estimation

Graph

Let the optical density of tubes T₁& T₂ be A and the corresponding protein concentration is B as shown in the graph.

Calculation

For  T₁& T₂

The optical density A of plasma corresponds to B mg of protein 0.1ml of plasma contains Bmg of protein

Therefore,

100ml of plasma will contain = 100 x B/ 0.1 mg of protein = Zmg of protein

Result: 

The amount of protein present in the given plasma sample = Zmg