ABTS
Encyclopedia
In biochemistry
, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS is chemical compound used to observe the reaction kinetics
of specific enzyme
s. A common use for it is in the enzyme-linked immunosorbent assay (ELISA
) to detect for binding of molecules to each other.
It is commonly used as a substrate
with hydrogen peroxide
for a peroxidase
enzyme or alone with blue multicopper oxidase
enzymes such as laccase
or bilirubin oxidase
. Its use allows the reaction kinetics of peroxidases themselves to be followed. In this way it also can be used to indirectly follow the reaction kinetics of any hydrogen peroxide
-producing enzyme, or to simply quantify the amount of hydrogen peroxide in a sample.
The formal reduction potentials for ABTS are high enough for it to act as an electron donor for the reduction of oxo species such as molecular oxygen and hydrogen peroxide, particularly at the less-extreme pH values encountered in biological catalysis. Under these conditions, the sulfonate groups are fully deprotonated and the mediator exists as a dianion.
This compound is chosen because the enzyme facilitates the reaction with hydrogen peroxide, turning it into a green and soluble
end-product. Its new absorbance maximum of 420 nm light
(ε = 3.6 × 104 M–1 cm–1) can easily be followed with a spectrophotometer, a common laboratory instrument.
It is sometimes used as part of a glucose
estimating reagent
when finding glucose concentrations of solutions such as blood serum
.
ABTS is also frequently used by the food industry and agricultural researchers to measure the antioxidant
capacities of foods. In this assay, ABTS is converted to its radical cation by addition of sodium persulfate
. This radical cation is blue in color and absorbs light at 734 nm. The ABTS radical cation is reactive towards most antioxidants including phenolics
, thiols and Vitamin C
. During this reaction, the blue ABTS radical cation is converted back to its colorless neutral form. The reaction may be monitored spectrophotometrically. This assay is often referred to as the Trolox equivalent antioxidant capacity
(TEAC) assay. The reactivity of the various antioxidants tested are compared to that of Trolox
, which is a water-soluble analog of vitamin E
.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS is chemical compound used to observe the reaction kinetics
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
of specific enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s. A common use for it is in the enzyme-linked immunosorbent assay (ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
) to detect for binding of molecules to each other.
It is commonly used as a substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
with hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
for a peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
enzyme or alone with blue multicopper oxidase
Multicopper oxidase
In molecular biology, multicopper oxidases are enzymes which oxidise their substrate by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear copper centre; dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two...
enzymes such as laccase
Laccase
Laccases are copper-containing oxidase enzymes that are found in many plants, fungi, and microorganisms. The copper is bound in several sites; Type 1, Type 2, and/or Type 3. The ensemble of types 2 and 3 copper is called a trinuclear cluster . Type 1 copper is available to action of solvents,...
or bilirubin oxidase
Bilirubin oxidase
In enzymology, a bilirubin oxidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are bilirubin and O2, whereas its two products are biliverdin and H2O....
. Its use allows the reaction kinetics of peroxidases themselves to be followed. In this way it also can be used to indirectly follow the reaction kinetics of any hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
-producing enzyme, or to simply quantify the amount of hydrogen peroxide in a sample.
The formal reduction potentials for ABTS are high enough for it to act as an electron donor for the reduction of oxo species such as molecular oxygen and hydrogen peroxide, particularly at the less-extreme pH values encountered in biological catalysis. Under these conditions, the sulfonate groups are fully deprotonated and the mediator exists as a dianion.
- ABTS–· + e– → ABTS2– E°′Reduction potentialReduction potential is a measure of the tendency of a chemical species to acquire electrons and thereby be reduced. Reduction potential is measured in volts , or millivolts...
= 0.67 V vs SHEStandard hydrogen electrodeThe standard hydrogen electrode , is a redox electrode which forms the basis of the thermodynamic scale of oxidation-reduction potentials... - ABTS + e– → ABTS–· E°′Reduction potentialReduction potential is a measure of the tendency of a chemical species to acquire electrons and thereby be reduced. Reduction potential is measured in volts , or millivolts...
= 1.08 V vs SHEStandard hydrogen electrodeThe standard hydrogen electrode , is a redox electrode which forms the basis of the thermodynamic scale of oxidation-reduction potentials...
This compound is chosen because the enzyme facilitates the reaction with hydrogen peroxide, turning it into a green and soluble
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
end-product. Its new absorbance maximum of 420 nm light
Light
Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...
(ε = 3.6 × 104 M–1 cm–1) can easily be followed with a spectrophotometer, a common laboratory instrument.
It is sometimes used as part of a glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
estimating reagent
Reagent
A reagent is a "substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs." Although the terms reactant and reagent are often used interchangeably, a reactant is less specifically a "substance that is consumed in the course of...
when finding glucose concentrations of solutions such as blood serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
.
ABTS is also frequently used by the food industry and agricultural researchers to measure the antioxidant
Antioxidant
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When...
capacities of foods. In this assay, ABTS is converted to its radical cation by addition of sodium persulfate
Sodium persulfate
Sodium persulfate is a chemical compound. It is a strong oxidizer. It is a severe irritant of skin, eyes, and respiratory system. It is almost non-hygroscopic and has particularly good ability to be stored for long time. It is easy and safe to handle...
. This radical cation is blue in color and absorbs light at 734 nm. The ABTS radical cation is reactive towards most antioxidants including phenolics
Polyphenol
Polyphenols are a structural class of natural, synthetic, and semisynthetic organic chemicals characterized by the presence of large multiples of phenol structural units...
, thiols and Vitamin C
Vitamin C
Vitamin C or L-ascorbic acid or L-ascorbate is an essential nutrient for humans and certain other animal species. In living organisms ascorbate acts as an antioxidant by protecting the body against oxidative stress...
. During this reaction, the blue ABTS radical cation is converted back to its colorless neutral form. The reaction may be monitored spectrophotometrically. This assay is often referred to as the Trolox equivalent antioxidant capacity
Trolox equivalent antioxidant capacity
Trolox equivalent antioxidant capacity measures the antioxidant capacity of a given substance, as compared to the standard, Trolox. Most commonly, antioxidant capacity is measured using the ABTS Decolorization Assay...
(TEAC) assay. The reactivity of the various antioxidants tested are compared to that of Trolox
Trolox
Trolox is Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant, like vitamin E, and is used in biological or biochemical applications to reduce oxidative stress or damage.Trolox equivalent antioxidant...
, which is a water-soluble analog of vitamin E
Vitamin E
Vitamin E is used to refer to a group of fat-soluble compounds that include both tocopherols and tocotrienols. There are many different forms of vitamin E, of which γ-tocopherol is the most common in the North American diet. γ-Tocopherol can be found in corn oil, soybean oil, margarine and dressings...
.