Biuret test
Encyclopedia
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 a violet
-colored complex in an alkaline solution. Several variants on the test have been developed.
The Biuret reaction can be used to assay 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
.
In spite of 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 bonds in the biuret molecule.
(KOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate
. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.
Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein
assay
, a colorimetric assay used to determine protein concentration
—such as UV-VIS at wavelength 540 nm (to detect the Cu2+ ion).
. 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 (malachite green
, Auramin O), resulting in further amplification of the signal.
Cu+ forms a deep purple complex with bicinchoninic acid
(BCA), which allows proteins in the range of 0.0005 to 2 mg/mL to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit.
Chemical test
In chemistry, a chemical test is a qualitative or quantitative procedure designed to prove the existence of, or to quantify, a chemical compound or chemical group with the aid of a specific reagent...
used for detecting the presence of peptide bonds. In the presence of peptides, a copper
Copper
Copper is a chemical element with the symbol Cu and atomic number 29. It is a ductile metal with very high thermal and electrical conductivity. Pure copper is soft and malleable; an exposed surface has a reddish-orange tarnish...
(II) ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
forms a violet
Violet (color)
As the name of a color, violet is synonymous with a bluish purple, when the word "purple" is used in the common English language sense of any color between blue and red, not including either blue or red...
-colored complex in an alkaline solution. Several variants on the test have been developed.
The Biuret reaction can be used to assay the concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume 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
Beer-Lambert law
In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...
.
In spite of its name, the reagent does not in fact contain biuret
Biuret
Biuret is a chemical compound with the chemical formula H2NCNHCNH2. It is the result of condensation of two molecules of urea and is a problematic impurity in urea-based fertilizers. This white solid is soluble in hot water....
((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide 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.Biuret reagent
The biuret reagent is made of potassium hydroxidePotassium hydroxide
Potassium hydroxide is an inorganic compound with the formula KOH, commonly called caustic potash.Along with sodium hydroxide , this colorless solid is a prototypical strong base. It has many industrial and niche applications. Most applications exploit its reactivity toward acids and its corrosive...
(KOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate
Potassium sodium tartrate
Potassium sodium tartrate is a double salt first prepared by an apothecary, Pierre Seignette, of La Rochelle, France. As a result the salt was known as Seignette's salt or Rochelle salt....
. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.
Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
, a colorimetric assay used to determine protein concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
—such as UV-VIS at wavelength 540 nm (to detect the Cu2+ ion).
Increasing the sensitivity of the biuret test
Cu+ is a strong reducing agent which can react for example with Mo(VI) in Folin-Ciocalteu's reagent to form molybdenum blueMolybdenum blue
Molybdenum blue is a term applied to:*reduced heteropolymolybdate complexes, polyoxometalates containing Mo, Mo, and a hetero atom such as phosphorus or silicon...
. 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 (malachite green
Malachite green
Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper...
, Auramin O), resulting in further amplification of the signal.
Cu+ forms a deep purple complex with bicinchoninic acid
Bicinchoninic acid
Bicinchoninic acid is a weak acid composed of two carboxylated quinoline rings.Bicinchoninic acid is most commonly employed by biochemists in the bicinchoninic acid assay, which is used to determine the total level of protein in a solution...
(BCA), which allows proteins in the range of 0.0005 to 2 mg/mL to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit.
See also
- Lowry protein assayLowry protein assayThe 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 measured using colorimetric techniques. It is...
- another method for estimating the concentration of protein in a solution