Biuret test

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Biuret test is used for detecting the presence of peptide bonds. It relies on the reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. It is possible to use the biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to Lambert-Beer's law.

The process can be done by adding an equal volume of 1% of KOH (potassium hydroxide) and few drops of 1 % of CuSO4 to the sample solution. If the solution turns purple, protein is present, 5-160 mg/ml can be determined.


[edit] Increasing the sensitivity of the biuret test

Cu+ is a strong reducing agent which can react for example with Mo(VI) in Folin-Ciocalteu's reagent to form molybdenum blue, a complex of 179410 Mo(V) and Mo(IV) ions. This is the basis of the Folin et al.[1] protein determination method, which can detect proteins in concentrations between 0.005 and 2 mg/ml. Molybdenum blue in turn can bind certain organic dyes (malachite green, Auramin O), resulting in further amplification[2] of the signal.

Cu+ also forms a bright pink complex with bicinchonic acid (BCA)[3], this allows proteins in the range of 0.0005 to 2 mg/ml to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit.

[edit] References

  1. ^ O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Mesurement With the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.
  2. ^ Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.
  3. ^ Smith, P.K. et al.: Measurement of protein using bicinchonic acid. Anal. Biochem. 150 (1985) 76-85.
  1. Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.

O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Mesurement With the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275

Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.

Smith, P.K. et al.: Measurement of protein using bicinchonic acid. Anal. Biochem. 150 (1985) 76-85.

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