Assay Genie’s non-radioactive, colorimetric fumarase assay is based on the reduction of the tetrazolium salt MTT in a NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm. The increase in absorbance at 565 nm is proportional to the enzyme activity.
For quantitative determination of fumarase enzyme activity.
- Fast and sensitive. Linear detection range: 0.4 to 70 U/L for 30 min reaction at 37°C.
- Convenient and high-throughput. Homogeneous “mix-incubate-measure” type assay. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
|Kit Includes||Assay Buffer: 10 mL Enzyme A: 120 µL NAD/MTT: 1 mL Enzyme B: 120 µL Substrate: 600 µL Calibrator: 1.5 mL|
|Kit Requires||Pipetting devices and accessories (e.g. multi-channel pipettor), clear flat- bottom 96-well plates, centrifuge tubes and plate reader|
|Method of Detection||OD565nm|
|Detection Limit||0.4 U/L|
|Samples||Biological samples (e.g. plasma, serum, erythrocytes, tissue and culture media.)|
|Protocol Length||40 min|
|Storage||Store all components at -20°C upon receiving.|
|Shelf Life||6 months|
FUMARASE (or fumarate hydratase) (EC 220.127.116.11) is an enzyme that catalyzes the reversible hydration/dehydration reaction of fumarate to malate. Fumarase exists in two isoforms: a cytosolic and mitochondrial form. In the citric acid cycle, it facilitates a transition step in the production of energy in the form of NADH. Fumarase deficiency in humans results in early brain development problems and is characterized by poor feeding, hypotonia, failure to thrive, etc.
Aliases for FH Gene
- Fumarate Hydratase
- EC 18.104.22.168
- Fumarate Hydratase, Mitochondrial
Entrez Gene Summary for FH Gene
The protein encoded by this gene is an enzymatic component of the tricarboxylic acid (TCA) cycle, or Krebs cycle, and catalyzes the formation of L-malate from fumarate. It exists in both a cytosolic form and an N-terminal extended form, differing only in the translation start site used. The N-terminal extended form is targeted to the mitochondrion, where the removal of the extension generates the same form as in the cytoplasm. It is similar to some thermostable class II fumarases and functions as a homotetramer. Mutations in this gene can cause fumarase deficiency and lead to progressive encephalopathy. [provided by RefSeq, Jul 2008]
UniProtKB/Swiss-Prot for FH Gene FUMH_HUMAN,P07954
Also acts as a tumor suppressor.
Protein details for FH Gene (UniProtKB/Swiss-Prot)
- Protein Symbol:P07954-FUMH_HUMAN
- Recommended name:Fumarate hydratase, mitochondrial
- Protein Accession:P07954
Secondary Accessions: B1ANK7
Protein attributes for FH Gene
- Size:510 amino acids
- Molecular mass:54637 Da
- Quaternary structure:Homotetramer.
- Miscellaneous: There are 2 substrate-binding sites: the catalytic A site, and the non-catalytic B site that may play a role in the transfer of substrate or product between the active site and the solvent. Alternatively, the B site may bind allosteric effectors (By similarity).
Post-translational modifications for FH Gene
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1. Saini, A. and Pratibha S. (2013) Infantile metabolic encephalopathy due to fumarase deficiency. Journal of child
neurology 28.4: 535-537.
2. Yogev, O, et al. (2010) Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA
damage response. PLoS biology 8.3: e1000328.
3. Rustin, P., et al. (1997) Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.
Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1361.2 : 185-197