Biuret test
The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colored coordination complexes in an alkaline solution.[1] Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.[2]
The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.
Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.
Contents |
Procedure [edit]
An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined. A peptide of a chain length of at least 3 amino acids is necessary for a significant, measurable color shift with these reagents.[3]
Biuret reagent [edit]
The Biuret reagent is made of potassium hydroxide (KOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate.[4] Potassium sodium tartrate[5] is added to complex and stabilize the cupric ions.The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.
Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein assay, a colorimetric assay used to determine protein concentration—such as UV-VIS at wavelength 540 nm (to detect the Cu2+ ion).
High sensitivity variants of the biuret test [edit]
Two major types of the biuret test are commonly applied in modern colorimetric analysis of peptides: the Bicinchoninic Acid (BCA) assay and the Modified Lowry assay. Both methods rely on the formation of a colored chelate complex of peptide bonds and cupric ions.
In the BCA test, Cu+ forms a deep purple complex with bicinchoninic acid (BCA),[6] which 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. The cupric ion coordinates to 4 peptide bonds, reducing it to a cuprous ion and allowing it to form a complex with BCA that absorbs around 540 nm, producing the signature violet color.[7] The BCA protein assay increases the sensitivity of the biuret test by a factor of around 100, and gives the important benefit of compatibility with samples that contain up to 5% surfactants. This is achieved by the chelation of bicinchoninic acid with the cuprous ion formed by the biuret reaction. This increases sensitivity as the water soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex.
The Modified Lowry protein assay utilizes a mechanism similar to the BCA protein assay; the protein is reacted with the copper(II) sulfate to produce the tetradentate copper complex, and a phosphomolybdic-phosphotungstic acid solution is added, in order to be reduced to a complex that absorbs very strongly at 650-750 nm.[8]
Cu+ is a reducing agent which can react for example with Mo(VI) in Folin-Ciocalteu's reagent to form molybdenum blue. In this way, proteins can be detected in concentrations between 0.005 and 2 mg/mL.[9] Molybdenum blue in turn can bind certain organic dyes such as malachite green and Auramin O, resulting in further amplification of the signal.[10]
See also [edit]
- Lowry protein assay - another method for estimating the concentration of protein in a solution
References [edit]
- ^ The reaction was first observed 1833: Ferdinand Rose (1833) "Über die Verbindungen des Eiweiss mit Metalloxyden" (On the compounds of albumin with metal oxides), Poggendorfs Annalen der Physik und Chemie, vol. 104, pages 132-142, doi:10.1002/andp.18331040512. It was independently rediscovered in 1857: G. von Piotrowski (1857) "Eine neue Reaction auf Eiweisskörper und ihre näheren Abkömmlinge" (A new reaction of proteins and their related derivatives) Sitzungsberichte der Kaiserliche Akademie der Wissenschaften in Wien, mathematisch-naturwissenschaftliche Classe (Proceedings of the Imperial Academy of Philosophies in Vienna, mathematical-natural sciences section), vol. 24, pages 335-337.
- ^ “Chemistry of Protein Assay” Thermo Scientific Protein Methods Library. www.piercenet.com
- ^ Fenk, C. J.; Kaufman, N.; and Gerbig, D. G. J. Chem. Educ. 2007, 84, 1676-1678.
- ^ Chemical Reagents
- ^ Chemical Reagents
- ^ Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.
- ^ Smith, P.K et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.
- ^ Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc.
- ^ O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement 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.
External links and notes [edit]
- Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.
- Chemical Reagents
