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Published on: August 17, 2019
Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P)
Andrew D Napper1, Sharmila Sivendran1
1Penn Center for Molecular Discovery, Institute for Medicine and Engineering, and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 1160 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104.
A new miniaturized fluorescent assay enables efficient screening of enzymes that use nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. This method accurately measures NADPH to NADP+ conversion in low volumes, minimizing false positives.
Area of Science:
- Biochemistry and Enzymology
- Assay Development
- Fluorescence Spectroscopy
Background:
- Enzymatic assays are crucial for drug discovery and understanding metabolic pathways.
- Monitoring cofactor conversion, such as nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+, is essential for characterizing enzyme activity.
- Existing methods may lack miniaturization, sensitivity, or robustness for high-throughput screening.
Purpose of the Study:
- To develop and validate a miniaturized fluorescent assay for monitoring NADPH to NADP+ conversion.
- To demonstrate the assay's utility in screening enzymes that utilize NADPH as a cofactor.
- To establish a protocol that minimizes false positive results from interfering compounds.
Main Methods:
- A miniaturized fluorescent assay was designed to measure the decrease in fluorescence emission at 460 nm.
- The assay monitors the turnover of NADPH to NADP+ over 90 minutes in low-volume 384-well plates.
- A secondary assay was implemented to identify and exclude compounds exhibiting inherent fluorescence at 460 nm.
Main Results:
- The developed assay successfully monitored NADPH consumption by enzymes.
- Assay validation demonstrated its reliability for enzyme screening.
- The follow-up assay effectively ruled out false positives caused by fluorescent artifacts.
Conclusions:
- A miniaturized, sensitive, and robust fluorescent assay for NADPH to NADP+ conversion has been established.
- This assay format is suitable for screening NADPH-dependent enzymes in a high-throughput manner.
- The protocol enhances the reliability of enzymatic screening by mitigating interference from fluorescent compounds.
