Dehydrogenases

Dehydrogenases are family of enzymes belonging to the group oxidoreductase. These enzymes catalyze the oxidation of a substrate by transferring one or more hydrides to an electron acceptor or coenzyme. Common electron acceptors, being considered as oxidizers of the substrate are usually nicotinamide adenine dinucleotide (NAD+/NADH), nicotinamide adenine dinucleotide phosphate (NADP+/NADPH), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

AAT Bioquest has developed a variety of optical assay kits either for detecting specific dehydrogenases targets such as Glucose-6-Phosphate Dehydrogenase and Lactate Dehydrogenase, or for general dehydrogenases based on the change of NAD+/NADH and NADP+/NADPH.

Glucose-6-Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-?-lactone, the first and rate-limiting step in the pentose phosphate pathway. It is critical metabolic pathway that supplies reducing energy to cells (such as erythrocytes) by maintaining the level of co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH), and for the production of pentose sugars. The production of NADPH is of great importance for tissues actively engaged in biosynthesis of fatty acids and/or isoprenoids, such as the liver, mammary glands, adipose tissue, and the adrenal glands. NADPH also maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage. Deficiencies in G6PD predispose individuals to non-immune hemolytic anemia.

Amplite™ Glucose-6-Phosphate Dehydrogenase Assays

Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit provides a simple, sensitive and rapid method for detecting G6PD in a variety of biological samples including serum, plasma, urine, and in cell culture samples. In the assay, G6PD activity is proportionally related to the concentration of NADPH that is specifically monitored using a fluorogenic NADPH sensor to yield a highly red fluorescence product. The signal can be monitored with a fluorescence microplate reader at Ex/Em = 540 nm/590 nm. The assay can detect G6PD activity from as little as 0.3 mU/mL in a 100 µL reaction volume.
Also available is our Amplite™ Colorimetric Glucose-6-Phosphate Dehydrogenase Assay kit. In the assay, G6PD activity is monitored by a chromogenic NADPH sensor. The signal can be read using an absorbance microplate reader at an absorbance ratio of Abs575 nm to Abs605 nm. The assay can detect as little as 3 mU/mL G6PD in a 100 µL reaction volume. For higher sensitivity, we recommond our Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit.

Figure 1. G6PD dose response was measured with Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.

Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is an oxidoreductase enzyme that catalyzes the interconversion of pyruvate and lactate. LDH is present in cytosol of a wide variety of organisms, including animals and plants. Cells release LDH into the bloodstream after tissue damage or red blood cell hemolysis. Since LDH is a fairly stable enzyme, it has been widely used to evaluate the presence of damage and toxicity of tissue and cells. Quantification of LDH has a broad range of applications. LDH is also elevated in certain pathological conditions such as cancer.
AAT Bioquest Amplite™ Lactate Dehydrogenase Assay Kits provide a simple and senstive method to measure either L-lactate dehydrogenase (L-LDH) or D-lactate dehydrogenase (D-LDH) activity in various types of samples. The assays are robust, readily adapted for a wide variety of applications that require the measurement of L-LDH or D-LDH.

Amplite™ Colorimetric Lactate Dehydrogenase Assays

Amplite™ Colorimetric D-Lactate Dehydrogenase Assay and Amplite™ Colorimetric L-Lactate Dehydrogenase Assay Kits provide a simple and quick absorbance-based method to measure lactate dehydrogenase activity in a broad scope of biological samples including serum, plasma, urine, as well as in cell culture samples. Both assays utilize and coupled-enzyme reaction in which LDH reduces NAD+ to NADH. The reduced NADH cofactor reacts with a chromogenic NADH sensor producing a stable color (?575 nm) that is directly proportional to LDH activity.
Assay Kit 13809 is specific for D-LDH and is suitable to detect as low as 3 mU/mL D-LDH activity in a 100 µL reaction volume. Assay Kit 13813 is specific for L-LDH and is suitable to detect as low as 3 mU/mL L-LDH activity in a 100 µL reaction volume.

Figure 2. D-LDH dose response was measured with Amplite™ Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.

Amplite™ Fluorimetric Lactate Dehydrogenase Assays

Amplite™ Fluorimetric D-Lactate Dehydrogenase Assay and Amplite™ Fluorimetric L-Lactate Dehydrogenase Assay Kits provide a highly sensitive fluorescence-based method to measure lactate dehydrogenase activity in a broad scope of biological samples including serum, plasma, urine, as well as in cell culture samples. Both assays utilize and coupled-enzyme reaction in which LDH reduces NAD+ to NADH. The reduced NADH cofactor reacts with a fluorogenic NADH sensor generating a strong signal (?590 nm) that is directly proportional to LDH activity.
Assay Kit 13808 is specific for D-LDH and is suitable to detect as low as 1 mU/mL D-LDH activity in a 100 µL reaction volume. Assay Kit 13812 is specific for L-LDH and is suitable to detect as low as 1 mU/mL L-LDH activity in a 100 µL reaction volume.

Figure 3. L-LDH dose response was measured with Amplite™ Fluorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.

Other Dehydrogenases

Additionally, changes in NAD/NADH and NADP/NADPH levels can be readily used to monitor the acitivty of dehydrogenases that utilize NAD/NADH or NADP/NADPH as a cofactor. The conventional NAD/NADH and NADP/NADPH assays are based on monitoring changes in NADH or NADPH absorption at 340 nm. The short UV wavelength of the traditional NAD/NADH and NADP/NADPH assays makes traditional methods suffer low sensitivity and high interference. Due to the weak absorption of NAD and NADH, the UV absorption method requires large sample sizes, making the same NAD and NADH measurement unpractical if the availability of samples is limited. AAT Bioquest has developed a full product line for detecting NAD, NADH, NADP and NADPH either spectrophotometrically or fluorimetrically.

Amplite™ NAD/NADH Assays

Amplite™ NAD/NADH Assay Kits provide a convenient and sensitive method to detect NAD and NADH. The NAD/NADH recycling enzyme mix in these kits recognizes NAD/NADH in an enzyme cycling reaction, which significantly improves the detection sensitivity and specificity. In addition, the assays have very low background interfence since the assays are performed in the red visible range. Working in this region considerably reduces interferences caused by autofluorescing biomoleculces within samples. There is no requirement to purify NAD/NADH from the sample mix.
Besides fluorimetric NAD/NADH assay kits, we offer 6 colorimetric assay kits for detecting NAD, NADH, NADP, and NADPH. These kits use longer wavelengths (?585 nm or ?635 nm) as their detection window. The longer absorption significantly reduces the interferences from other absorbing species that generally absorbs light in the UV and short visible wavelengths.

Figure 4. NADH dose responses were measured with Amplite™ Colorimetric NAD/NADH Assay Kit in a 96-well white wall/clear bottom plate using a NOVOStar (BMG Labtech) microplate reader. As low as 300 nM (30 pmol/well) of NADH can be detected with 1 hour incubation time (n=3).

Amplite™ NADP/NADPH Assays

Amplite™ NADP/NADPH Assay Kits provide a convenient method for sensitive detection of NADP and NADPH. There is no need to purify NADP/NADPH from the sample mix. Amplite™ Fluorimetric Total NADP/NADPH Assay or Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kits are recommended if higher sensitivity is required. The fluorescence signal can be detected at Ex/Em = 540/590 nm.

Figure 5. Total NADP and NADPH, and their extract dose responses were measured with Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit in a 96-well black plate using a Gemini microplate reader (Molecular Devices). The signals were acquired at Ex/Em = 540/590 nm (cut off at 570 nm) 30 minutes after adding 75 µL of NADPH reaction mixture.

Product Ordering Information

Table 1. Ordering Info for LDH Assays Products

Cat#Product NameUnit Size
13808Amplite™ Fluorimetric D-Lactate Dehydrogenase (LDH) Assay Kit200 Tests
13809Amplite™ Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit200 Tests
13812Amplite™ Fluorimetric L-Lactate Dehydrogenase (LDH) Assay Kit200 Tests
13813Amplite™ Colorimetric L-Lactate Dehydrogenase (LDH) Assay Kit200 Tests

Table 2. Ordering Info for NAD(P)/NAD(P)H Assays Products

Cat#Product NameUnit Size
15257Amplite™ Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*400 Tests
15258Amplite™ Colorimetric Total NAD and NADH Assay Kit400 Tests
15259Amplite™ Fluorimetric Total NADP and NADPH Assay Kit *Red Fluorescence*400 Tests
15260Amplite™ Colorimetric Total NADP and NADPH Assay Kit400 Tests
15261Amplite™ Fluorimetric NADH Assay Kit *Red Fluorescence*400 Tests
15262Amplite™ Fluorimetric NADPH Assay Kit *Red Fluorescence*400 Tests
15263Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*250 Tests
15264Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*250 Tests
15265ReadiUse™ NADPH Regenerating Kit1000 Tests
15266ReadiUse™ NADP Regenerating Kit200 Tests
15271Amplite™ Colorimetric NADH Assay Kit400 Tests
15272Amplite™ Colorimetric NADPH Assay Kit400 Tests
15273Amplite™ Colorimetric NAD/NADH Ratio Assay Kit250 Tests
15274Amplite™ Colorimetric NADP/NADPH Ratio Assay Kit250 Tests
15275Amplite™ Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*400 Tests
15276Amplite™ Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*400 Tests
15280Amplite™ Fluorimetric NAD Assay Kit *Blue Fluorescence*200 Tests
15281Amplite™ Fluorimetric NADP Assay Kit *Blue Fluorescence*200 Tests
15290Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit100 Tests
15291Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit100 Tests
15295Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit *Deep Red Fluorescence*100 Tests
15296Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit *Deep Red Fluorescence*100 Tests

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