The Lowry protein assay is a
biochemical
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology an ...
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 of a ...
for determining the total level of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
in a
solution
Solution may refer to:
* Solution (chemistry), a mixture where one substance is dissolved in another
* Solution (equation), in mathematics
** Numerical solution, in numerical analysis, approximate solutions within specified error bounds
* Soluti ...
. 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 named for the biochemist
Oliver H. Lowry who developed the
reagent
In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 300,000 times.
Mechanism
The method combines the reactions of copper ions with the
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 under
alkaline
In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...
conditions (the
Biuret test
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 , where ...
) with the
oxidation
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
of aromatic
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
residues. The Lowry method is based on the reaction of Cu
+, produced by the oxidation of peptide bonds, with
Folin–Ciocalteu reagent The Folin–Ciocâlteu reagent (FCR) or Folin's phenol reagent or Folin–Denis reagent, is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric ''in vitro'' assay of phenolic and polyphenolic antioxidants, also called t ...
(a mixture of
phosphotungstic acid
Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula . It forms hydrates . It is normally isolated as the ''n'' = 24 hydrate but can be desiccated to the hexahydrate (''n'' = 6). EPTA is the nam ...
and
phosphomolybdic acid
Phosphomolybdic acid is the heteropolymetalate with the formula . It is a yellow solid, although even slightly impure samples have a greenish coloration. It is also known as dodeca molybdophosphoric acid or PMA, is a yellow-green chemical compound ...
in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves
reduction of the Folin–Ciocalteu reagent and
oxidation
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
of
aromatic
In chemistry, aromaticity is a chemical property of cyclic ( ring-shaped), ''typically'' planar (flat) molecular structures with pi bonds in resonance (those containing delocalized electrons) that gives increased stability compared to satur ...
residues (mainly
tryptophan
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α- carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
, also
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 non-essential amino acid with a polar side group. The word "tyrosine" is from the Gr ...
). Experiments have shown that
cysteine
Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile.
When present as a deprotonated catalytic residue, sometime ...
is also reactive to the reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry assay. The result of this reaction is an intense blue molecule known as heteropolymolybdenum Blue. The concentration of the reduced Folin reagent (heteropolymolybdenum Blue) is measured by
absorbance
Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative lo ...
at 660 nm.
As a result, the total concentration of protein in the sample can be deduced from the concentration of tryptophan and tyrosine residues that reduce the Folin–Ciocalteu reagent.
The method was first proposed by Lowry in 1951. The
bicinchoninic acid assay
The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company, now part of Thermo Fisher Scientific, is a biochemical assay for determining the total concentration of prot ...
and the
Hartree–Lowry assay are subsequent modifications of the original Lowry procedure.
See also
*
Biuret test
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 , where ...
*
Bradford protein assay
The Bradford protein assay was developed by Marion M. Bradford in 1976. It is a quick and accurate spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composi ...
References
* Walker, J. M. (2002). The protein protocols handbook. Totowa, N.J: Humana Press.
External links
A simplification of the protein assay method of Lowry et al. which is more generally applicable
Biochemistry detection reactions
Protein methods
Analytical chemistry
Reagents for biochemistry
Protein Estimation using Lowry Method in Acube Lifesciences
{{Analytical reagents