Biuret Reaction
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chemistry Chemistry is the scientific study of the properties and behavior of matter. It is a physical science within the natural sciences that studies the chemical elements that make up matter and chemical compound, compounds made of atoms, molecules a ...
, the biuret test (IPA: , ), also known as Piotrowski's test, is a
chemical test In chemistry, a chemical test is a qualitative property, qualitative or Quantitative property, quantitative procedure designed to identify, quantify, or characterise a chemical compound or substituent, chemical group. Purposes Chemical testing m ...
used for detecting the presence of at least two
peptide bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...
s in a molecule. In the presence of peptides, a
copper Copper is a chemical element; it has symbol Cu (from Latin ) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pinkish-orang ...
(II)
ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
forms
mauve Mauve ( ; ) is a pale purple color named after the mallow flower (French: ). The first use of the word ''mauve'' as a color was in 1796–1798 according to the ''Oxford English Dictionary'', but its use seems to have been rare before 1859. ...
-colored
coordination complex A coordination complex is a chemical compound consisting of a central atom or ion, which is usually metallic and is called the ''coordination centre'', and a surrounding array of chemical bond, bound molecules or ions, that are in turn known as ' ...
es in an
alkaline In chemistry, an alkali (; from the Arabic word , ) is a basic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of a soluble base has a pH greater than 7.0. The ...
solution. The reaction was first observed in 1833. In
Poland Poland, officially the Republic of Poland, is a country in Central Europe. It extends from the Baltic Sea in the north to the Sudetes and Carpathian Mountains in the south, bordered by Lithuania and Russia to the northeast, Belarus and Ukrai ...
, the biuret test is also known as Piotrowski's test in honor of the Polish physiologist who independently rediscovered it in 1857. 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 In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', '' molar concentration'', '' number 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 The Beer–Bouguer–Lambert (BBL) extinction law is an empirical relationship describing the attenuation in intensity of a radiation beam passing through a macroscopically homogenous medium with which it interacts. Formally, it states that the ...
. Despite its name, the reagent does not in fact contain
biuret Biuret ( ) is a chemical compound with the chemical formula . It is a white solid that is soluble in hot water. A variety of organic derivatives are known. The term "biuret" also describes a family of organic compounds with the chemical formula , ...
. 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 Tris, or tris(hydroxymethyl)aminomethane, or known during medical use as tromethamine or THAM, is an organic compound with the formula (HOCH2)3CNH2. It is extensively used in biochemistry and molecular biology as a component of buffer solutions ...
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.


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 Copper(II) sulfate is an inorganic compound with the chemical formula . It forms hydrates , where ''n'' can range from 1 to 7. The pentahydrate (''n'' = 5), a bright blue crystal, is the most commonly encountered hydrate of copper(II) sulfate, whi ...
. If the solution turns purple, it contains protein. 5–160 mg/ 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.


Biuret reagent

The biuret reagent is made of
sodium hydroxide Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula . It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly corrosive base (chemistry), ...
(NaOH) and hydrated
copper(II) sulfate Copper(II) sulfate is an inorganic compound with the chemical formula . It forms hydrates , where ''n'' can range from 1 to 7. The pentahydrate (''n'' = 5), a bright blue crystal, is the most commonly encountered hydrate of copper(II) sulfate, whi ...
, together with
potassium sodium tartrate Potassium sodium tartrate tetrahydrate, also known as Rochelle salt, is a double salt of tartaric acid first prepared (in about 1675) by an apothecary, , of La Rochelle, France. Potassium sodium tartrate and monopotassium phosphate were the fi ...
, the latter of which is added to
chelate Chelation () is a type of bonding of ions and their molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These l ...
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. The reagent is commonly used in the biuret
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
assay An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity ...
, a
colorimetric Colorimetry is "the science and technology used to quantify and describe physically the human color perception". It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color p ...
test used to determine protein
concentration In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', '' molar concentration'', '' number concentration'', ...
by UV/VIS spectroscopy at wavelength 540 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 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 The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be ...
, Cu+ is oxidized back to Cu2+ by MoVI in the Folin–Ciocalteu reagent, which forms
molybdenum blue Molybdenum blue is a term applied to: *reduced heteropolymolybdate complexes, polyoxometalates containing Mo(V), Mo(VI), and a hetero atom such as phosphorus or silicon *reduced isopolymolybdate complexes, polyoxometalates containing Mo(V), Mo(VI) ...
(MoIV).
Tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a conditionally essential amino acid with a polar side group. The word "tyrosine" is ...
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 can in turn bind certain organic dyes such as
malachite green Malachite green is an organic compound that is used as a dyestuff and controversially as an antimicrobial in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Despite its name the dye is ...
and Auramine O, resulting in further amplification of the signal.Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.


References


External links and notes

* Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.
Chemical Reagents
{{Organic reactions Biochemistry detection reactions Protein methods biüret testi