D2322
Diaphorase from Clostridium kluyveri
recombinant, expressed in E. coli, Animal-component free
Synonym(s):
Diaphorase, Lipoamide Dehydrogenase, Lipoyl Dehydrogenase
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About This Item
recombinant
expressed in E. coli
description
Histidine-Tagged
form
solid
specific activity
>=30 units/mg protein (biuret)
storage temp.
−20°C
Application
Diaphorase is used in various assays to oxidate β-NADH or β-NADPH in the presence of an electron acceptor. Diaphorase from Clostridium Kluyveri has been used for semiquinone generation during benzoquinone reduction.
Biochem/physiol Actions
Diaphorase catalyzes the oxidation of either β-NADH or β-NADPH in the presence of an electron acceptor such as methylene blue or 2,6-dichlorophenolindophenol. Diaphorases which are specific for either β-NADH or β-NADPH are known. The pig heart enzyme of Straub seems to have native diaphorase (β-NADH specific) as well as lipoic and lipoamide dehydrogenase activities. It is reported to be a single protein. However, Massey reports that diaphorase is probably a denatured lipoamide dehydrogenase. Pre-incubation of the pig heart preparation with Cu2+ reduces the lipoamide dehydrogenase activity and proportionately increases the β-NADH diaphorase activity. In our laboratory, we have demonstrated this copper effect to some degree on the pig heart enzyme, but no appreciable effect was observed on the Clostridium kluyveri or torula yeast preparations. The lipoamide dehydrogenase:diaphorase ratio is a measure of the denaturation.
Physical form
Supplied as a lyophilized powder containing trehalose.
Other Notes
One unit of diaphorase will oxidize 1.0 micromole of beta-NADH per minute at pH 7.5 at 25 degrees C, with the corresponding reduction of the appropriate electron acceptor.
The name "Diaphorase" has been loosely applied to several enzymes which catalyze the oxidation of either β-NADH or β-NADPH in the presence of an electron acceptor such as methylene blue or 2,6-dichlorophenolindophenol. Many different assay procedures and "units" are used.
Diaphorases which are specific for either β-NADH or β-NADPH are known. The pig heart enzyme of Straub seems to have native diaphorase (β-NADH specific) as well as lipoic and lipoamide dehydrogenase activities. It is reported to be a single protein. However, Massey reports that "diaphorase" is probably a denatured lipoamide dehydrogenase. Pre-incubation of the pig heart preparation with Cu2+ reduces the lipoamide dehydrogenase activity and proportionately increases the β-NADH diaphorase activity. In our laboratory, we have demonstrated this copper effect to some degree on the pig heart enzyme, but no appreciable effect was observed on the Clostridium kluyveri or torula yeast preparations. The lipoamide dehydrogenase:diaphorase ratio is a measure of the denaturation.
Diaphorases which are specific for either β-NADH or β-NADPH are known. The pig heart enzyme of Straub seems to have native diaphorase (β-NADH specific) as well as lipoic and lipoamide dehydrogenase activities. It is reported to be a single protein. However, Massey reports that "diaphorase" is probably a denatured lipoamide dehydrogenase. Pre-incubation of the pig heart preparation with Cu2+ reduces the lipoamide dehydrogenase activity and proportionately increases the β-NADH diaphorase activity. In our laboratory, we have demonstrated this copper effect to some degree on the pig heart enzyme, but no appreciable effect was observed on the Clostridium kluyveri or torula yeast preparations. The lipoamide dehydrogenase:diaphorase ratio is a measure of the denaturation.
Storage Class
13 - Non Combustible Solids
wgk
WGK 3
flash_point_f
Not applicable
flash_point_c
Not applicable
Regulatory Information
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