Oxidases

Oxidases are enzymes that catalyze various oxidation-reduction reactions that typically involve the transfer of a hydrogen (H) molecule from a substrate to an oxygen (O) molecule to form water (H2O) and/or hydrogen peroxide (H2O2) as a by-product. Oxidases also have the potential to catalyze a diversity of oxidation reactions and can interact with amines (amino acid oxidases), alcohols (in carbs), influence oxidative ring closure, and may play active roles in decarboxylation and hydroxylation reactions. A number of species of oxidases exist, and each hold a separate, vital, part in biochemistry and physiology.

Common Oxidases

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NADPH Oxidase

NADPH oxidase reactions may produce the superoxide anion, O2-, the precursor to a number of reactive oxygen species (ROS) that are essential in killing many types of bacteria and other microorganisms. Because of the critical function NADPH plays in host defense, mutations in the molecular makeup of the enzyme may result in hereditary diseases, specifically chronic granulomatous disease.

Seven NADPH oxidase isoforms exist, termed NOX1-5, DUOX1, and DUOX2, that differ in their association with variable proteins, accumulation in certain tissues, intracellular localization, ROS formation, and regulation. NADH oxidase, on the other hand, is a flavoenzyme that uses flavin adenine dinucleotide (FAD) as a cofactor to transfer electrons between molecules and has been most readily studied in yeasts, bacteria, and other microbiota.

Cytochrome C Oxidase

Cytochrome c oxidase (commonly, CO, CCO or COX) is located in the inner mitochondrial membrane and functions as the terminal constituent of the electron transport chain. CCO forms water from molecular oxygen, which allows for the production of ATP, thereby aiding in the establishing mitochondrial membrane potential.

Monoamine Oxidase

Monoamine oxidases (MAO) are involved in removing various neurotransmitters including norepinephrine, serotonin, and dopamine from the brain. They can be categorized into two types; MAO-A preferentially oxidizes serotonin and is located in the brain and neurons, while MAO-B prefers to bind to phenethylamines and locates in the glia and astrocytes.

Xanthine Oxidase

Xanthine oxidases (XO) are necessary for the conversion of hypoxanthine to xanthine, and later uric acid which becomes excreted by the kidneys downstream.

Lysyl Oxidase

Lysyl oxidases (LO) are a class of copper-dependent amine oxidase that play a critical role in the creation of connective tissue matrices through enabling the crosslinking of collagen and elastin. LO dysregulation may in fact relate to many fibrotic diseases like dermatolysis and type V Ehlers-Danlos syndrome.

L-glutamate Oxidase

L-glutamate oxidase (LGOX) catalyzes the oxidative deamination of the enzyme into α-ketoglutarate (AKG), ammonia, and H2O2. AKG is a crucial molecule in the citric acid cycle, and determines the overall rate at which stored energy is released in an organism.

Tools

Overview of Assay Principles & Procedures

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Colorimetric/Fluorometric Assays

Many colorimetric as well as fluorometric, commercially available, kits exist that can quantitatively measure the extent of oxidase activity. Analysis can be performed easily using a spectrophotometer, microplate, or auto-analyser. Kits can be adapted to automation, allowing for high-throughput capabilities, offer quick experimental test times and are cost-effective.



For MAO isoforms, MAO-A and MAO-B react with p-tyramine, which in turn forms H2O2. As one unit of MAO catalyzes the formation of one µmole of H2O2 per minute under most assay conditions, enzyme activity can be fluorometrically determined using an excitation/emission (Ex/Em) ratio of 530/585 nm. Some modern kits use alternative fluorescent substrates, with varying Ex/Em ratios and sensitivities as needed by experimental requirements. For colorimetric kits, horseradish peroxidase (HRP) reacts with H2O2 to produce a red-pink colored product. Samples can be read at a specific absorbance and values are proportional to H2O2 levels, representative of amine oxidase levels, present within samples. Clorgyline, a specific inhibitor of MAO-A, and Pargyline, a specific inhibitor of MAO-B, are frequently included in kits to differentiate between the enzyme species.

Colorimetric and fluorometric kits for other oxidases (XO, LOX, LGOX, etc.) in principle, work similarly. As the enzymatic activity of oxidases may be linked to many biological processes, these kits can provide insight on a number of biochemical functions. NADPH and NADH oxidase kits have also been cited for mechanistic studies of various cancers, screening of anticancer drugs, and evaluating the effects between ROS and metabolism. Additionally, CO kits have been commonly used to determine the levels of active mitochondria within a sample.

ELISA

Enzyme-linked immunosorbent assay (ELISA) is another colorimetric means of in vitro quantitation of oxidase activity. ELISAs offer flexibility in that serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids can be used as starting material.

Though traditional ELISAs may be used, typically sandwich ELISAs are recommended as many oxidases characteristically have more than two possible epitopes. Sandwich ELISA techniques use an HRP conjugated reagent to react with the antibody of the desired oxidase. After unbound conjugates are removed, 3,3',5,5'-Tetramethylbenzidine (TMB), or other chromogenic HRP substrate such as ABTS or ReadiUse™ Stayright™ Purple quantifies the HRP reaction.

Only wells that contain sufficient amounts of the oxidase will produce a colored product (blue, typically), which changes to yellow after adding the stop solution. Upon analysis, the intensity of the yellow is directly proportional to the amount of oxidase in each well. Fluorescent and chemiluminescent substrates also exist, and are typically more sensitive, with the disadvantage of being at a higher price point.

Histochemistry/Immunohistochemistry Techniques

Histochemical and immunohistochemical staining techniques (HC and IHC) may also be used to assess enzyme activity. These methods provide the ability to map and quantify the differences in oxidase activity in discrete tissue and/or sample regions. HC and IHC are widely versatile, modifiable, and can use a number of staining techniques from immunofluorescence to metallic ions. HC, in general, stains tissues and cellular components, while IHC can selectively identify antigens within a cell through the use of antibodies.

HC and IHC are commonly multiplexed together, and analytical techniques may involve multi-spectral imaging and/or densitometric analysis. In this way oxidase activity may be measured in particular regions of a sample, as well as overall for the entire sample, using light, fluorescent, or even electron microscopy. One commonly analyzed oxidase using these methods is CCO oxidase (COX) due to its relationship with metabolism.

Bioluminescent Assays

Bioluminescent assays may be preferred as they can be easily adapted to high throughput screening, are more sensitive than fluorescent and colorimetric techniques, and can accurately measure the binding constants of particular substrates and inhibitors. Methods are low-cost, rapid, reproducible, and can be miniaturized for samples that have very small quantities of cells. In the presence of the target oxidase, a reductase provided with the assay will reduce a proluciferin substrate to form luciferin. Luciferin can be quantified using a detection reagent, and the luminescent signal produced is proportional to the amount of the oxidase in the sample. Bioluminescent assays exist, namely, for MAO, CCO, NADH and NADPH oxidases.

Product Ordering Information

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Resources

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Monoamine Oxidases (MAOs) as Privileged Molecular Targets in Neuroscience: Research Literature Analysis
Biochemistry, Xanthine Oxidase
Lysyl oxidase: properties, regulation and multiple functions in biology
Glucose Oxidase
NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology
Structural characterization of L-glutamate oxidase from Streptomyces sp. X-119-6
Cytochrome C Oxidase