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

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, res ...

in a solution (0.5 μg/mL to 1.5 mg/mL), similar to

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 ...

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 composit ...

or biuret reagent. The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using

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 ...

techniques.

Mechanism

A stock BCA solution contains the following ingredients in a highly alkaline solution with a pH 11.25:

bicinchoninic acid Bicinchoninic acid is a weak acid composed of two carboxylated quinoline rings. Bicinchoninic is an organic compound with the formula (C9H5NCO2H)2. The molecule consists of a pair of quinoline rings, each bearing a carboxylic acid group. Its s ...

, sodium carbonate, sodium bicarbonate,

sodium tartrate Sodium tartrate (Na2C4H4O6) is a salt used as an emulsifier and a binding agent in food products such as jellies, margarine, and sausage casings. As a food additive, it is known by the E number E335. Because its crystal structure captures a ve ...

, and

copper(II) sulfate Copper(II) sulfate, also known as copper sulphate, 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 h ...

pentahydrate. The BCA assay primarily relies on two reactions. First, the

peptide bonds 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 ...

in protein

reduce Reduction, reduced, or reduce may refer to: Science and technology Chemistry * Reduction (chemistry), part of a reduction-oxidation (redox) reaction in which atoms have their oxidation state changed. ** Organic redox reaction, a redox react ...

Cu²⁺ ions from the copper(II) sulfate to Cu¹⁺ (a temperature dependent reaction). The amount of Cu²⁺ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid

chelate Chelation is a type of bonding of ions and 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 ligands are ...

with each Cu¹⁺

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 conve ...

, forming a purple-colored complex that strongly absorbs light at a

wavelength In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats. It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, t ...

of 562 nm. The bicinchoninic acid Cu¹⁺ complex is influenced in protein samples by the presence of cysteine/cystine, tyrosine, and tryptophan side chains. At higher temperatures (37 to 60 °C), peptide bonds assist in the formation of the reaction complex. Incubating the BCA assay at higher temperatures is recommended as a way to increase assay sensitivity while minimizing the variances caused by unequal amino acid composition. The amount of protein present in a solution can be quantified by measuring the

absorption spectra Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating ...

and comparing with protein solutions of known concentration.

References

* *

External links

OpenWetWare

BCA assay chemistry
{{DEFAULTSORT:Bicinchoninic Acid Assay Biochemistry methods Chemical tests de:Bicinchoninsäure

Mechanism

See also

References

External links