NADH Oxidase（NOX）is widely found in the animals, plants, microorganisms and cultured cells, which can directly oxidize NADH to NAD+ in the presence of oxygen, and reduce blue DCPIP to colorless DCPIP. The activity of NOX can be calculated by measuring the reduction rate of blue DCPIP at 600 nm.
Isocitrate dehydrogenase (IDH) is one of the inverters in the tricarboxylic acid cycle and plays an important role in energy metabolism, amino acid and vitamin synthesis. The cofactors of this enzyme include two kinds of NAD+ and NADP+, which are located in different parts of the cell respectively. In eukaryotic cells, NAD-dependent isocitrate dehydrogenase mainly exists in mitochondria. Under the activation of the activator, IDH converts isocitrate into α-ketoglutaric acid. Meanwhile, NAD+ is reduced to NADH, which under the action of hydrogen transmitter, transfer electrons to WST-8 to produce the yellow product. The activity of NAD-IDH can be calculated by measuring the change of absorbance value at 450 nm.
NAD-MDH catalyzes the conversion of substrate malic acid and NAD to oxaloacetic acid and NADH. NADH makes WST-8 orange under the action of electron coupling agent. The activity of NAD-MDH can be calculated by measuring the change of absorbance value at 450 nm.
Detect total content of NAD+ and NADH Ethanol generates acetaldehyde under the action of enzyme. Meanwhile, NAD+ is reduced to NADH, NADH, under the action of hydrogen transmitter, transfer electrons to WST-8 to produce the yellow product, which has a characteristic absorption peak at 450 nm. Therefore, the total content of NAD+ and NADH can be quantified by measure the OD value at 450 nm. Detect NADH After treating sample, heat at 60oC water bath for 30 min. the NAD+ of the sample is decomposed and only NADH remains. NADH reduces WST-8 to form yellow product, and the amount of NADH is determined by measure the OD value at 450 nm. Detect NAD+ and NAD+/NADH The content of NAD+ and the ratio of NAD+/NADH in the sample can be obtained according to the total content of NAD+ and NADH obtained of the first two steps as well as the separate content of NADPH. Note: NADP+ and NADPH have no effect on the determination results.
NADPH Oxidase (NAO) is a membrane protein mainly found in mammalian neutrophils, plant cells and filamentous fungal cells, and was initially discovered in phagocytic cell membranes. NAO can oxidize NADPH to NAD+ while generating superoxide anions, and it is one of the main sources of reactive oxygen species (ROS) in the body. NAO is involved in many important physiological processes, including host defense, post-translational modification of proteins, cell signal transduction, gene expression regulation and cell differentiation. The detection principle of this kit is as follows: NAO catalyzes the substrate to produce product, which react with chromogenic agent to form chromogenic substance. It has the maximum absorption at 450 nm. The enzyme activity of NAO is calculated by measuring the OD value at 450 nm.
Isocitrate dehydrogenase converts isocitrate to α- ketoglutaric acid, while converting NADP+ to NADPH. Under the action of electron coupling agents, the generated NADPH has a characteristic absorption peak at 450 nm. The activity of NADP-IDH can be calculated by measuring the change of absorbance value at 450 nm.
NADP-MDH catalyzes the conversion of malic acid and NADP+ to oxaloacetic acid and NADPH. NADPH makes WST-8 orange under the action of electron coupling agent. The activity of NADP-MDH can be calculated by measuring the change of absorbance value at 450 nm.
Detect total content of NADP+ and NADPH: Glucose 6-phosphate (G6P) is oxidized to 6-phosphate gluconolactone (6-PG) by glucose-6-phosphate dehydrogenase (G6PDH), and NADP+ is reduced to NADPH during this reaction. NADPH, under the action of 1-mPMS, transfer electrons to WST-8 to produce the yellow product, which has a characteristic absorption peak at 450 nm. Therefore, the total content of NADP+ and NADPH can be quantified by measure the OD value at 450 nm. Detect NADPH: After treating sample, heat at 60℃ water bath for 30 min. the NADP+ of the sample is decomposed and only NADPH remains. NADPH reduces WST-8 to form Formazan, and the amount of NADPH is determined by measure the OD value at 450 nm. Detect NADP+ and NADP+/NADPH: The content of NADP+ and the ratio of NADP+/NADPH in the sample can be obtained according to the total content of NADP+ and NADPH obtained of the first two steps as well as the separate content of NADPH.
Na+K+-ATPase decomposes ATP to produce ADP and Phosphorus, and calculates the activity of Na+K+-ATPase by measuring the content of phosphorus.
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