Miniaturized High‐Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P)

Andrew D. Napper1, Sharmila Sivendran2

1 Nemours Center for Childhood Cancer Research, Wilmington, Delaware, 2 GlaxoSmithKline, Collegeville, Pennsylvania
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100155
Online Posting Date:  June, 2011
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This unit describes a miniaturized fluorescence assay that monitors the conversion of NADPH to NADP+. The same assay format may also be used to measure NADH to NAD+ conversion. Examples of assay development and validation results are presented to illustrate the use of this protocol to screen an enzyme that consumes NADPH as a cofactor during conversion of substrate to a reduced product. Enzymatic assays are carried out in low‐volume 384‐well plates, in which the turnover of NADPH is monitored by the decrease in fluorescence emission at 460 nm between an initial measurement and a second reading after 90 min. A follow‐up assay is used to rule out false‐positive artifacts arising from compounds that fluoresce at 460 nm. Curr. Protoc. Chem. Biol. 3:81‐97 © 2011 by John Wiley & Sons, Inc.

Keywords: NADPH detection; NADH detection; fluorescence; high‐throughput screening; oxidoreductase; enzyme assay; fluorescent artifact

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • NADPH (see recipe)
  • Assay buffer (see recipe)
  • Target enzyme(s): For the study used as an example here, the M. tb rhamnosyl biosynthetic enzymes dTDP‐6‐deoxy‐D‐xylo‐4‐hexulose 3,5‐epimerase (RmlC) and dTDP‐6‐deoxy‐L‐lyxo‐4‐hexulose reductase (RmlD) were cloned and expressed in E.coli and purified by Michael McNeil and co‐workers (Ma et al., ); these enzymes are not commercially available
  • Substrates: TDP‐6‐deoxy‐D‐xylo‐hexopyranosid‐4‐ulose (TDP‐KDX) was synthesized enzymatically and stored frozen at a concentration of 1 mg/ml in 50 mM MOPS buffer (Sigma), pH 7.4, at −80°C as previously described (Sivendran et al., ); this substrate is not commercially available
  • Positive control inhibitor: thymidine diphosphate (TDP; Sigma)
  • Analytical‐grade dimethyl sulfoxide (DMSO; Fisher or VWR), anhydrous
  • Test compounds: Store dissolved in DMSO (e.g., at 10 mM stock concentration) in 384‐well polypropylene plates (room temperature storage is recommended if the compounds are to be reused within a period of less than one week; for longer term storage, compounds stocks should be frozen at <−20°C)
  • Pipetting workstation equipped with 384‐tip MDT pipetting head and with pintool consisting of 384 pins with nominal transfer volume of 100 nl, e.g., a JANUS from Perkin Elmer or equivalent (earlier model known as Evolution EP3) (Rudnicki and Johnston, )
  • Compound dilution plates: Polypropylene V‐bottom 384‐well plates (Greiner Bio‐One, cat. no. 781280)
  • Assay plates: Black low‐volume 384‐well plate (Corning, cat. no. 3676)
  • Plate reader capable of reading fluorescence in 384‐well plates (e.g., an EnVision multimode plate reader from Perkin Elmer)
  • GraphPad Prism (or equivalent): for graphing data and curve fitting for IC 50 calculation
  • Multichannel pipettor
  • 25°C incubator
  • Reagent dispenser (e.g., a Multidrop‐384 reagent dispenser from Thermo Scientific; see Rudnicki and Johnston, )
  • 500‐µl microcentrifuge tubes
  • Microsoft Excel, OpenHTS (CeuticalSoft), or ActivityBase (IDBS) (or equivalent): for percent inhibition calculations, and evaluation of datasets and selection of hits
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Literature Cited

   Batchelor, R.H. and Zhou, M. 2002. A resorufin‐based fluorescent assay for quantifying NADH. Anal. Biochem. 305:118‐119.
   Batchelor, R.H. and Zhou, M. 2004. Use of cellular glucose‐6‐phosphate dehydrogenase for cell quantitation: Applications in cytotoxicity and apoptosis assays. Anal. Biochem. 329:35‐42.
   Bergmeyer, H.U. and Bernt, E. 1963. Lactate dehydrogenase. In Methods of Enzymatic Analysis. (H.U. Bergmeyer, ed.) pp. 574‐579. Academic Press, London.
   Fersht, A. 1985. Enzyme Structure and Mechanism, 2nd edition. Freeman, New York.
   Ma, Y., Stern, R.J., Scherman, M.S., Vissa, V.D., Yan, W., Jones, V.C., Zhang, F., Franzblau, S.G., Lewis, W.H., and McNeil, M.R. 2001. Drug targeting Mycobacterium tuberculosis cell wall synthesis: Genetics of dTDP‐rhamnose synthetic enzymes and development of a microtiter plate‐based screen for inhibitors of conversion of dTDP‐glucose to dTDP‐rhamnose. Antimicrob. Agents Chemother. 45:1407‐1416.
   Moran, J.H. and Schnellmann, R.G. 1996. A rapid beta‐NADH‐linked fluorescence assay for lactate dehydrogenase in cellular death. J. Pharmacol. Toxicol. Methods 36:41‐44.
   Rudnicki S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc.Chem. Biol. 1:43‐54.
   Segel, I. 1975. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady‐State Enzyme Systems. Wiley, New York.
   Sivendran, S., Jones, V., Sun, D., Wang, Y., Grzegorzewicz, A.E., Scherman, M.S., Napper, A.D., McCammon, J.A., Lee, R.E., Diamond, S.L., and McNeil, M. 2010. Identification of triazinoindol‐benzimidazolones as nanomolar inhibitors of the Mycobacterium tuberculosis enzyme TDP‐6‐deoxy‐d‐xylo‐4‐hexopyranosid‐4‐ulose 3,5‐epimerase (RmlC). Bioorg. Med. Chem. 18:896‐908.
Internet Resources
  Results and analysis from screening 265,000 compounds against RmlC/D enzymes.
  Dose response testing following RmlC/D HTS and hit confirmation by curve fitting and IC50 determination.
  Dose response testing to eliminate fluorescent compounds as false positives following RmlC/D HTS.
  Section of the Assay Guidance Manual that describes how to measure Km.
  Section of the Assay Guidance Manual that describes how to determine IC50 values.
  Section of the Assay Guidance Manual that describes the relationship of enzyme inhibitor IC50 to substrate concentration and how this relates to the mechanism of inhibition.
  GraphPad Prism Regression Guide, which provides a highly detailed discussion and many examples of nonlinear regression.
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