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D5540

Sigma-Aldrich

Diaphorase from Clostridium kluyveri

lyophilized powder, 3.0-20.0 units/mg protein (biuret)

Synonym(s):

Diaphorase, Lipoamide Dehydrogenase, Lipoyl Dehydrogenase

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About This Item

CAS Number:
9001-18-7
Enzyme Commission number:
1.8.1.4 (BRENDA, IUBMB)
EC Number:
232-587-6
MDL number:
MFCD00130947
UNSPSC Code:
12352204
NACRES:
NA.54
Pricing and availability is not currently available.

biological source

bacterial (Clostridium kluyveri)

Quality Level

200

form

lyophilized powder

specific activity

3.0-20.0 units/mg protein (biuret)

shipped in

wet ice

storage temp.

−20°C

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Application

Diaphorase from Clostridium kluyveri, or Lipoyl dehydrogenase, has been used in a study to assess the protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism. Lipoyl dehydrogenase has also been used in a study to investigate the redox regulation of tyrosine nitration and 3-nitrotyrosine reduction by antioxidants.
Methylene blue (MB)-containing polyacrylamide nanoparticle platforms (NPs) were tested in solution with diaphorase from Clostridium kluyveri and the cofactor NADH. this test done done to check whether the encapsulation of MB in NPs could prevent the reduction of MB, and thus protect its photodynamic effectiveness.[1] The enzyme from Sigma has been used along with aldehyde dehydrogenase to construct a biosensor for acetaldehyde by immobilization.[2] It has also been used in the reoxidation of NADP to NADPH. This reaction simultaneously catalyzed the conversion of resazurin into the highly fluorescent resorufin, and also allowed the detection of minute amounts of NAADP. NAADP was initially converted to NADP and NADPH by other enzymes.[3]

Packaging

Sold on the basis of native diaphorase units.

Other Notes

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.

Unit Definition

One unit of either "diaphorase" or "lipoyl" dehydrogenase will oxidize 1.0 μmole of β-NADH per min at pH 7.5 at 25 °C, with the corresponding reduction of 2,6-dichlorophenolindophenol

inhibitor

Product No.
Description
Pricing

C0400

Carmustine, ≥98%

Storage Class Code

11 - Combustible Solids

WGK

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US), Eyeshields, Gloves

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Certificates of Analysis (COA)

Lot/Batch Number
0000418419
0000418419
0000401434
0000401433
0000353359

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Bioelectronic sniffer with a diaphragm flow-cell for acetaldehyde vapor.
Mitsubayashi K, et al.
Sensors and Actuators B, Chemical, 95(1-3), 303-308 (2003)
Daniel Demus et al.
Glycobiology, 32(3), 230-238 (2021-12-24)
Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N-glycans. The excellent diagnostic
Wei Tang et al.
Biochemical and biophysical research communications, 369(2), 579-583 (2008-02-27)
The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs).
Kirti Rani et al.
Indian journal of biochemistry & biophysics, 43(2), 98-104 (2006-09-08)
3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed
Hauh-Jyun Candy Chen et al.
Chembiochem : a European journal of chemical biology, 9(2), 312-323 (2007-12-29)
Covalent modifications of proteins by endogenous reactive nitrogen oxide species lead to cytotoxic effects that are implicated in diseases associated with chronic infections and inflammation. Tyrosine nitration is a major post-translational modification of proteins by reactive nitrogen oxide species. Recent
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Questions

  1. I am looking to reconstitute the solid but cannot find any information on what to use for this. Any advice? Thanks.

    1 answer

    1. Prior to being assayed, this enzyme is reconstituted at a concentration of 1 mg/ml in cold 200 mM Tris-HCl buffer containing 294 mM Potassium Chloride, 0.54 mM Riboflavin 5'-monophosphate, and 0.025% (w/v) BSA, pH 7.5.

      (Detailed information could be found in the enzymatic assay(link below):
      https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/diaphorase.pdf)

      Helpful?

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