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Enzyme Activity Measurement for Oxidoreductases Using Spectrophotometric Assays



Oxidoreductases are a class of enzymes that catalyze the transfer of electrons from one molecule to another. The molecule that gives the electrons are called the electron donor or reductant, and the one receiving the electrons are called the electron acceptor or oxidant. Oxidoreductase enzymes play an important role in metabolic process. They can be found in both aerobic and anaerobic pathways, including glycolysis, TCA cycle, oxidative phosphorylation, and amino acid metabolism. To complete their functions, the oxidoreductase enzymes utilize redox cofactors. Common redox cofactors include FAD (flavin adenine dinucleotide), Fd (ferredoxin) FMN (flavin mononucleotide), NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), Coenzyme B, Coenzyme Q, etc. For example, the enzyme glyceraldehydes-3-phosphate dehydrogenase reduce NAD+ to NADH in glycolysis. While NADH is re-oxidized to NAD+ in the oxidative phosphorylation pathway to maintain the proper re-dox state of the cell. Pyruvate is produced during glycolysis, which is then reduced to lactate through anaerobic glycolysis using NADH as the reductant. The redox cofactors bear one or more aromatic or unsaturated cyclic chemical groups in their structures. As a result, the cofactors strongly absorb UV or visible light in the reduced or oxidized form, or both. This makes the redox cofactors perfect reporters to the spectrophotometric enzyme assays. 

Oxidorecuctases can be categorized into different subtypes, including oxidases and dehydrogenases. Oxidase enzymes use the molecular oxygen as the acceptor of hydrogen or electrons. To the contrary, dehydrogenase enzymes transfer hydrogen to NAD, NADP, or a flavin in order to oxidize a substrate. Other oxidoreductases include reductases, peroxidases, hydroxylases, and oxygenases. Reductases could also be oxidases since most redox reactions are reversible. The direction of the redox reaction depends both thermodynamics and kinetics. Peroxidases catalyze the decomposition of hydrogen peroxide, which is a natural defensive process against environmental invaders. Hydroxylases incorporate hydroxyl groups into organic compounds, while oxygenases add oxygen from molecular oxygen to the substrates.

Creative Enzymes proudly offers spectrophotometric enzyme assays in quantification of activity levels for all types of oxidoreductase enzymes:

As a large and important family of enzymes, oxidoreductases are used widely in the pharmaceutical industry for syntheses of amino acids, steroids, and other molecules; in the chemical industry for production of specialty chemicals; in clinical diagnosis and other analytical applications; and in other applications such as material modification and pollution control.

Enzyme Activity Measurement for Oxidoreductases Using Spectrophotometric Assays Figure: The structure of a human cytochrome P450 reductase (EC 1.6.2.4) showing cofactors NADPH (blue) and FAD (magenta), and a prosthetic group FMN (yellow).
Reference: Jin, Y.; Chen, M.; Penning, T.; Miller, W. Biochem. J. 2015, 468 (1), 25.


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