View source for Biuret test

{{short description|Chemical test for detecting peptide bonds}}
[[File:Biuret Test 2.jpg|thumb|alt=A footbal containing air clear mauve solution|The characteristic color of a positive biuret test]]

In [[chemistry]], the '''biuret test''' (IPA: {{IPAc-en|ˌ|b|aɪ|y|ə|ˈ|r|ɛ|t}}, {{IPAc-en|ˈ|b|aɪ|y|ə| ˌ|r|ɛ|t}}<ref>{{Cite web|title=Definition of biuret {{!}} Dictionary.com|url=https://www.dictionary.com/browse/biuret|access-date=2021-03-11|website=www.dictionary.com|language=en|archive-date=2021-05-11|archive-url=https://web.archive.org/web/20210511192032/https://www.dictionary.com/browse/biuret|url-status=live}}</ref>), also known as '''Piotrowski's test''', is a [[chemical test]] used for detecting the presence of at least two [[peptide bond]]s in a molecule. In the presence of peptides, a [[copper]](II) [[ion]] forms [[mauve]]-colored [[coordination complex]]es in an [[alkaline]] solution. The reaction was first observed in 1833.<ref>{{cite journal |last=Rose |first=Ferdinand |year=1833 |title=Über die Verbindungen des Eiweiss mit Metalloxyden |trans-title=On the compounds of albumin with metal oxides |url=https://books.google.com/books?id=cS04AAAAMAAJ&dq=%C3%9Cber+die+Verbindungen+des+Eiweiss+mit+Metalloxyden&pg=PA132 |url-status=live |journal=Poggendorff's Annalen der Physik und Chemie |language=de |location=Leipzig, Germany |publisher=J.A. Barth |volume=104 |issue=5 |pages=132-[https://books.google.com/books?id=cS04AAAAMAAJ&dq=%C3%9Cber+die+Verbindungen+des+Eiweiss+mit+Metalloxyden&pg=PA142 142] |bibcode=1833AnP...104..132R |doi=10.1002/andp.18331040512 |oclc=1481215 |archive-url=https://web.archive.org/web/20220509171524/https://books.googleusercontent.com/books/content?req=AKW5QaczDZVhISDmQKgm_PVrsejQphRrDcCKCjNyGLAgz2BqePQ-Ex44TPul5kPhgne2lkPoWV-5v7KKg6dBV8CWNB5cYelO12Ucu_AntFLJeGgOScnB8dD-oLhbq6T8BPYnHwkXGZylfreNsZXPCZsH3W779E8x0hgE3DJ8jDYyXg6Tr7JYZcIIe912GBP4o_nb5xgtP53wQsUUqcjrXFY6LUWtcIn7SI-6ys4_ZG-ue7gpgpWL0MFxMwq__OeHSaFNHwTAEWvLNsJl14qyKxZewWMaTuOVHw |archive-date=9 May 2022}}</ref> In [[Poland]], the biuret test is also known as Piotrowski's test in honor of the Polish physiologist {{ill|Gustaw Piotrowski|pl}} who independently rediscovered it in 1857.<ref>{{cite journal |last=Piotrowski |first=G. |year=1857 |title=Eine neue Reaction auf Eiweisskörper und ihre näheren Abkömmlinge |trans-title=A new reaction of proteins and their related derivatives |url=https://books.google.com/books?id=QcE5AAAAcAAJ |url-status=live |journal=Sitzungsberichte der Kaiserliche Akademie der Wissenschaften, Mathematisch-naturwissenschaftliche Classe (Meeting Reports of the Imperial Academy of Sciences, Mathematical-scientific Class) |language=de |location=Vienna |volume=24 |pages=335–337 |oclc=166037616 |archive-url=https://web.archive.org/web/20220509172648/https://books.googleusercontent.com/books/content?req=AKW5Qaed1JhNZFVVvlPCHmGGDKkhqQBQIfBQVX1mQvlj2VjyVAnXc2djFUhc4ob6CC8LO4gg85WIolKBXvK83_ovN6ITIMjPTeJ7daX3IFcIFGs2aOBXTNzzONXOD9bkVi-AcBjl1zXQ32vJuJC1OeycYtqnf0zICg7FFNU2lxwMg6GvybSVwoqCavKRL5ZsF7-ST1jc73fcg_wB-XzPpCsvz59aqhnVr9ZXfP3X2ka8p0pba6RL1JQMNlEZKusGFy1VeRS5LXXMq8ort8olkiO-CtuTgkSeMQxBdiw2dvXH_4KNVm6cheg |archive-date=9 May 2022}}</ref> Several variants on the test have been developed, such as the [[Bicinchoninic acid assay|BCA test]] and the Modified Lowry test.<ref>{{cite web|title=Chemistry of Protein Assay|publisher=Thermo Fisher Scientific Protein Methods Library|url=https://www.thermofisher.com/in/en/home/brands/thermo-scientific/pierce-protein-biology.html#/legacy=www.piercenet.com#:~:text=BCA%20Protein%20Assay|url-status=live|access-date=2022-05-08|archive-date=2022-03-24|archive-url=https://web.archive.org/web/20220324180528/https://www.thermofisher.com/in/en/home/brands/thermo-scientific/pierce-protein-biology.html#/legacy=www.piercenet.com}}</ref>

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&nbsp;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]] {{chem2|[(H2N\sCO\s)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.<ref>{{Cite book |last1=Ninfa |first1=Alexander |url=https://books.google.com/books?id=263UDwAAQBAJ |title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology |last2=Ballou |first2=David |last3=Benore |first3=Marilee |publisher=Wiley |year=2009 |isbn=978-0470087664 |pages=111 |oclc=1288381941 |access-date=2022-05-09 |archive-date=2022-05-09 |archive-url=https://web.archive.org/web/20220509173057/https://www.google.co.in/books/edition/Fundamental_Laboratory_Approaches_for_Bi/263UDwAAQBAJ?hl=en |url-status=live }}</ref>

==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, it contains protein. 5–160&nbsp;mg/[[millilitre|mL]] can be determined. Peptides with the correct length of at least 3 amino acids are necessary for a significant, measurable colour shift with these reagents.<ref>Fenk, C. J.; Kaufman, N.; and Gerbig, D. G. J. Chem. Educ. 2007, 84, 1676-1678.</ref>

==Biuret reagent==
The '''biuret reagent''' is made of [[sodium hydroxide]] (NaOH) and hydrated [[copper(II) sulfate]], together with [[potassium sodium tartrate]],<ref>{{Cite web |url=http://faculty.mansfield.edu/bganong/biochemistry/reagents.htm |title=Chemical Reagents |access-date=2010-01-30 |archive-url=https://web.archive.org/web/20100213004815/http://faculty.mansfield.edu/bganong/biochemistry/reagents.htm |archive-date=2010-02-13 |url-status=dead }}</ref> the latter of which 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 characteristic color. This is found with dipeptides.<ref>{{Cite journal|last1=Datta|first1=S. P.|last2=Leberman|first2=R.|last3=Rabin|first3=B. R.|date=1959|title=The chelation of metal ions by dipeptides and related substances. Part 5.—Cupric complexes of sarcosyl and leucyl ligands|url=http://xlink.rsc.org/?DOI=TF9595502141|journal=Trans. Faraday Soc.|language=en|volume=55|pages=2141–2151|doi=10.1039/TF9595502141|issn=0014-7672|access-date=2020-08-29|archive-date=2022-05-09|archive-url=https://web.archive.org/web/20220509173024/https://pubs.rsc.org/en/content/articlelanding/1959/TF/TF9595502141|url-status=live|url-access=subscription}}</ref>

The reagent is commonly used in the biuret [[protein]] [[assay]], a [[colorimetric]] test used to determine protein [[concentration]] by [[UV/VIS spectroscopy]] at wavelength 540&nbsp;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<sup>+</sup> formed during the biuret reaction reacts further with other reagents, leading to a deeper color.

In the [[Bicinchoninic acid assay|BCA test]], Cu<sup>+</sup> forms a deep purple complex with [[bicinchoninic acid]] (BCA),<ref>Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.</ref> which absorbs around 562&nbsp;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&nbsp;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<sup>+</sup> is oxidized back to Cu<sup>2+</sup> by Mo<sup>VI</sup> in the [[Folin–Ciocalteu reagent]], which forms [[molybdenum blue]] (Mo<sup>IV</sup>). [[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&nbsp;mg/mL.<ref>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.</ref> Molybdenum blue can in turn bind certain organic dyes such as [[malachite green]] and [[Auramine O]], resulting in further amplification of the signal.<ref>Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.</ref>
== References ==
{{reflist}}

==External links and notes==
{{Commons category}}

* Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.
* [https://web.archive.org/web/20100213004815/http://faculty.mansfield.edu/bganong/biochemistry/reagents.htm Chemical Reagents]

{{Protein methods}}
{{Analytical reagents}}
{{Organic reactions}}

[[Category:Biochemistry detection reactions]]
[[Category:Protein methods]]
[[Category:Chemical tests|biüret testi]]