α-Ketoglutarate Assay Based on Fluorescence Quenching by NADH

Sharma, Ashutosh; Quantrill, Nigel S. M.

doi:10.1021/bp960027f

Cited by 7 publications

(4 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…31 The authors describe this as "substrate-induced quenching" (SIQ) based on a light-induced redox reaction. We intend to present a more detailed analysis and possible applications of this method in a separate publication.…”

Section: Brightness Change Due To Quenching By a Small Molecule Analytementioning

confidence: 99%

“…After completing the experiments reported here, we found that a similar phenomenon as the NADH-and light-dependent quenching of resorufin has been reported for the structurally related dye thionine. 31 The authors describe this as "substrate-induced quenching" (SIQ) based on a light-induced redox reaction. We intend to present a more detailed analysis and possible applications of this method in a separate publication.…”

Section: Brightness Change Due To Quenching By a Small Molecule Analytementioning

confidence: 99%

See 1 more Smart Citation

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

et al. 2003

View full text Add to dashboard Cite

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. ( Journal of Biomolecular Screening 2003:19-33)

show abstract

Section: Brightness Change Due To Quenching By a Small Molecule Analytementioning

confidence: 99%

Section: Brightness Change Due To Quenching By a Small Molecule Analytementioning

confidence: 99%

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

et al. 2003

View full text Add to dashboard Cite

show abstract

“…However, data reported elsewhere by us and by Sharma have verified that the reaction product of excited thionine and NADH and NADPH is NAD + and NADP + , respectively, by its ability to act as an electron acceptor in NAD + or NADP + specific enzyme reactions. [35][36][37] These results indicate that the quenching of thionine is at least partially due to an excited-state redox process that results in a net electron transfer from NADH or NADPH to thionine.…”

Section: Reaction Mechanismmentioning

confidence: 86%

Photochemical Coenzyme Regeneration in an Enzymatically Active Optical Material

et al. 2004

View full text Add to dashboard Cite

The photoinduced electron transfer between immobilized thionine and the dinucleotide enzyme cofactors NADH and NADPH in a SiO 2 sol-gel matrix is reported. The electron-transfer quenching of thionine luminescence is used to monitor the rate of NADPH oxidation. Using Stern-Volmer quenching curves, the quenching rates in the silica matrix are 1 to 2 orders of magnitude smaller than those in solution. The rate constants for oxidation of NADPH by thionine were measured to be 9.8((2.9) × 10 -3 s -1 in solution and 8.8((1.0) × 10 -4 s -1 in the gel. Within the silica matrix, the photoinduced oxidation of NADPH is combined with the enzymatic reaction of isocitrate dehydrogenase, which uses the oxidized cofactor, NADP + , as an electron acceptor in the oxidation of isocitrate. The encapsulated isocitrate dehydrogenase is active with a Michaelis-Menten constant, K M , of 3 µM and a k cat of 0.67 µM/s per mg enzyme . Because optical sensors use NADPH fluorescence as an indicator of the presence and relative concentration of enzyme substrate, the successful demonstration of photoinduced regeneration of NADP + makes possible continuous monitoring by the family of dehydrogenase enzymes.

show abstract

“…Consequently, this method was applied to measure ethanol 3 and glucose levels 7 and to monitor the activity of α-ketoglutarate. 8 However, the method had not been used in miniaturized, HTScompatible format.…”

Section: Introductionmentioning

confidence: 99%

Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption

Vázquez¹,

Ashman²,

Ramón³

et al. 2006

SLAS Discovery

View full text Add to dashboard Cite

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

show abstract

α-Ketoglutarate Assay Based on Fluorescence Quenching by NADH

Cited by 7 publications

References 2 publications

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

Photochemical Coenzyme Regeneration in an Enzymatically Active Optical Material

Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption

Contact Info

Product

Resources

About