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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.〔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, . It was independently rediscovered in 1857 by a Polish physiologist: G. 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.〕 Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.〔“Chemistry of Protein Assay” Thermo Scientific Protein Methods Library. http://www.piercenet.com〕
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.
==Procedure==
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.〔Fenk, C. J.; Kaufman, N.; and Gerbig, D. G. J. Chem. Educ. 2007, 84, 1676-1678.〕
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