Biuret test
In
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 named so because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.
In this assay, the copper(II) binds with nitrogen atoms present in the peptides of proteins. In a secondary reaction, the copper(II) is reduced to copper(I). Buffers, such as Tris and ammonia interfere with this assay, therefore rendering this assay inappropriate for protein samples purified from ammonium sulfate precipitation. Due to its insensitivity and little interference by free amino acids, this assay is most useful for whole tissue samples and other sources with high protein concentration.[5]
Procedure
An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide) followed by a few drops of aqueous
Biuret reagent
The Biuret reagent is made of
The reagent is commonly used in the biuret
High sensitivity variants of the biuret test
Two major modifications of the biuret test are commonly applied in modern colorimetric analysis of peptides: the bicinchoninic acid (BCA) assay and the Lowry assay. In these tests, the Cu+ formed during the biuret reaction reacts further with other reagents, leading to a deeper color.
In the BCA test, Cu+ forms a deep purple complex with bicinchoninic acid (BCA),[9] which absorbs around 562 nm, producing the signature mauve color. The water-soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex, increasing the sensitivity of the biuret test by a factor of around 100: the BCA assay allows to detect proteins in the range of 0.0005 to 2 mg/mL). Additionally, the BCA protein assay gives the important benefit of compatibility with substances such as up to 5% surfactants in protein samples.
In the Lowry protein assay Cu+ is oxidized back to Cu2+ by MoVI in the Folin–Ciocalteu reagent, which forms molybdenum blue (MoIV). Tyrosine residues in the protein also form molybdenum blue under these circumstances. In this way, proteins can be detected in concentrations between 0.005 and 2 mg/mL.[10] Molybdenum blue in turn can bind certain organic dyes such as malachite green and Auramine O, resulting in further amplification of the signal.[11]
References
- ^ "Definition of biuret | Dictionary.com". www.dictionary.com. Archived from the original on 2021-05-11. Retrieved 2021-03-11.
- from the original on 9 May 2022.
- from the original on 9 May 2022.
- ^ "Chemistry of Protein Assay". Thermo Fisher Scientific Protein Methods Library. Archived from the original on 2022-03-24. Retrieved 2022-05-08.
- OCLC 1288381941. Archivedfrom the original on 2022-05-09. Retrieved 2022-05-09.
- ^ Fenk, C. J.; Kaufman, N.; and Gerbig, D. G. J. Chem. Educ. 2007, 84, 1676-1678.
- ^ "Chemical Reagents". Archived from the original on 2010-02-13. Retrieved 2010-01-30.
- from the original on 2022-05-09. Retrieved 2020-08-29.
- ^ Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.
- ^ 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
- Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.
- Chemical Reagents