Biuret test - wikipedia Biuret test The characteristic color of a positive biuret test The biuret test ( Piotrowski 's 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. Several variants on the test have been developed, such as the BCA test and the Modified Lowry test. 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 ( ( H N - CO - ) NH ). 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 nitrogens 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. Contents 1 Procedure 2 Biuret reagent 3 High sensitivity variants of the biuret test 4 References 5 External links and notes 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 copper ( II ) sulfate. If the solution turns purple, protein is present. 5 -- 160 mg / mL can be determined. Peptides with the chain length of at least 3 amino acids are necessary for a significant, measurable colour shift with these reagents. Biuret reagent The Biuret reagent is made of sodium hydroxide ( NaOH ) and hydrated copper ( II ) sulfate, together with potassium Chemical Reagents Potassium sodium tartrate is added to chelate and thus stabilize the cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or tetra dentate chelation with the peptide nitrogen produces the `` biuret '' color. This is found with dipeptides ( Datta, S.P., Leberman, R., and Rabin, B.R., Trans. Farad. Soc. ( 1959 ), 55, 2141. ) The reagent is commonly used in the biuret protein assay, a colorimetric test used to determine protein concentration by UV / VIS spectroscopy at wavelength 550 nm. 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 ), which absorbs around 562 nm, producing the signature violet 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 Cu by Mo in Folin - Ciocalteu 's reagent, which forms molybdenum blue ( Mo ). 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. Molybdenum blue in turn can bind certain organic dyes such as malachite green and Auramine O, resulting in further amplification of the signal. In Poland, the Biuret Test is more commonly called as the Piotrowski Test, in honor of the Polish toxological chemist, Jerzy Kazimierz Piotrowski ( b. 1926 ) of Lodz. References Jump up ^ Charles William Hale : Domestic Science. Cambridge : Cambridge University Press, 1915, s. 229, seria : Cambridge technical series. Jump up ^ 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 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. Jump up ^ `` Chemistry of Protein Assay '' Thermo Scientific Protein Methods Library. http://www.piercenet.com Jump up ^ Ninfa, Alexander ; Ballou, David ; Benore, Marilee ( 2009 ). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Wiley. p. 111. ISBN 978 - 0470087664. Jump up ^ Fenk, C.J. ; Kaufman, N. ; and Gerbig, D.G.J. Chem. Educ. 2007, 84, 1676 - 1678. Jump up ^ Chemical Reagents Jump up ^ Smith, P.K. et al. : Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 ( 1985 ) 76 - 85. Jump up ^ 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. Jump up ^ 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 Proteins : key methods of study Experimental Protein purification Green fluorescent protein Western blot Protein immunostaining Protein sequencing Gel electrophoresis / Protein electrophoresis Protein immunoprecipitation Peptide mass fingerprinting / Protein mass spectrometry Dual - polarization interferometry Microscale thermophoresis Chromatin immunoprecipitation Surface plasmon resonance Isothermal titration calorimetry X-ray crystallography Protein NMR Cryo - electron microscopy Freeze - fracture electron microscopy Bioinformatics Protein structure prediction Protein -- protein docking Protein structural alignment Protein ontology Protein -- protein interaction prediction Assay Enzyme assay Protein assay Secretion assay Display techniques Bacterial display mRNA display Phage display Ribosome display Yeast display Super-resolution microscopy Photoactivated localization microscopy Vertico SMI Analytical reagents and tests Metals Aluminon Xylenol orange Sugars, fats, and proteins Sugars & Starches Benedict 's reagent ( reducing sugars etc ) Aniline acetate test ( pentoses ) Starch indicator Molisch 's test ( carbs ) Proteins & amino acids Biuret test ( proteins ) Bicinchoninic acid assay ( concentration ) Bradford protein assay ( concentration ) Lowry protein assay ( total protein ) Pierce Protein Assay ( total protein ) Tollens ' reagent ( aldehyde & ketones ) Fehling 's solution ( aldehyde & ketones ) Van Slyke determination ( amino acids ) Sakaguchi test ( arginine ) Fats Sudan stain ( lipids ) Alcohols Benedict 's reagent ( aldehyde & ketones etc ) Denigés ' reagent Lucas ' reagent ( types of alcohols ) Drugs Dille -- Koppanyi reagent ( barbiturates etc ) Ehrlich 's reagent ( indoles, etc ) Froehde reagent ( opioids ) Gallic acid reagent ( drug precursor ) Liebermann reagent ( street drugs ) Mandelin reagent ( ketamine ) Marquis reagent ( MDMA, opiates, etc ) Mecke reagent Pill testing ( general ) Simon 's reagent Zimmermann reagent ( Benzodiazepines ) Zwikker reagent ( barbiturates ) Salicylate testing other Dragendorff 's reagent ( alkaloids ) Fenton 's reagent ( creates free radicals ) Folin -- Ciocalteu reagent ( antioxidants ) Murexide test ( caffeine etc ) Melzer 's reagent ( fungi ) Marquis reagent ( various ) Nitrate test Nitrite test Luminol ( blood ) Pesticide detection kit Fecal coliform detection Prion detection kit Food portal Drink portal Category Commons Retrieved from `` https://en.wikipedia.org/w/index.php?title=Biuret_test&oldid=801511462 '' Categories : Protein methods Chemical tests Hidden categories : All articles with unsourced statements Articles with unsourced statements from April 2017 Talk Contents About Wikipedia Čeština Deutsch Euskara Français 한국어 Հայերեն Italiano Қазақша Magyar Nederlands 日本 語 Polski Português Русский සිංහල Svenska தமிழ் ไทย Українська 中文 Edit links This page was last edited on 20 September 2017, at 03 : 04. 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Question: how can you tell by using this test that a substance contains protein
Answer: biuret test