Biuret Protein Assay

The bond between the amino group and the carboxyl acid group on adjacent amino acids in a protein is a peptide bond. When the Biuret reagent (1 percent solution of copper sulfate) is added to a solution containing peptide bonds, the solution turns a violet color. The violet color is a positive test for the presence of protein. The more intense the color, the greater the number of peptide bonds that react.

Principle:

Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.

Equipment:

In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 450 nm. Glass or polystyrene (cheap) cuvettes may be used.

methodology

Reagent

A formula for biuret reagent is (per liter final volume) 9 gm Sodium potassium tartrate (f.w. 282.22), 3 gm Copper sulfate x 5 H2O (f.w. 249.68), 5 gm Potassium iodide (166.0), all dissolved in order in 400 ml 0.2 M NaOH (f.w. 40.0) before bringing to final volume. The volume can be scaled up or scaled down of course. Discard if a black precipitate forms.

Assay

1. Volumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
2. Warm up the spectrophotometer 15 min. before use.
3. Prepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction coefficient. Using 5 ml color reagent to 1 ml sample a recommended range is 0.5 to 20 mg protein.
4. Prepare a reference tube with 1 ml buffer.
5. If possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated.
6. Use 1 ml sample per assay tube
7. Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
8. Read at 550 nm.

Analysis

Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

Comments

The color is stable, but all readings should be taken within 10 min. of each other. As with most assays, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups will give high or low readings, respectively.
For Bovine serum albumin we typically obtain a linear relationship between absorbance and amount protein over a range of 0.5 to 20 mg protein. The assay has not been reliable for amounts below 0.5 mg, however the actual sensitive range may extend beyond the upper limit.

References

• Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949.
• Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957.
• Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940.
• Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269.
• Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.