Skip to main content
SpringerLink
Log in

Extraction and Measurement of NAD(P)+ and NAD(P)H

  • Protocol
  • First Online:
Pseudomonas Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1149))

Abstract

Nicotinamide adenine dinucleotides are critical redox-active substrates for countless catabolic and anabolic reactions. Ratios of NAD+ to NADH and NADP+ to NADPH are therefore considered key indicators of the overall intracellular redox potential and metabolic state. These ratios can be measured in bulk conditions using a highly sensitive enzyme cycling-based colorimetric assay (detection limit at or below 0.05 μM or 1 pmol) following a simple extraction procedure involving solutions of acid and base. Special considerations are necessary to avoid measurement artifacts caused by the presence of endogenous redox-active metabolites, such as phenazines made by diverse Pseudomonas species (see Chapter 25).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. de Graef MR, Alexeeva S, Snoep JL, Teixeira de Mattos MJ (1999) The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J Bacteriol 181:2351–2357

    Article  Google Scholar 

  2. Hoek JB, Rydström J (1988) Physiological roles of nicotinamide nucleotide transhydrogenase. Biochem J 254:1–10

    Article  CAS  Google Scholar 

  3. Harrison D, Chance B (1970) Fluorimetric technique for monitoring changes in the level of reduced nicotinamide nucleotides in continuous cultures of microorganisms. Appl Microbiol 19:446–450

    Article  CAS  Google Scholar 

  4. Chen F, Xia Q, Ju L-K (2003) Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(P)H fluorescence. Appl Environ Microbiol 69:6715–6722

    Article  CAS  Google Scholar 

  5. London J, Knight M (1966) Concentrations of nicotinamide nucleotide coenzymes in micro-organisms. J Gen Microbiol 44:241–254

    Article  CAS  Google Scholar 

  6. Hung YP, Albeck JG, Tantama M, Yellen G (2011) Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor. Cell Metab 14:545–554

    Article  CAS  Google Scholar 

  7. Bernofsky C, Swan M (1973) An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 53:452–458

    Article  CAS  Google Scholar 

  8. Price-Whelan A, Dietrich LEP, Newman DK (2007) Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14. J Bacteriol 189:6372–6381

    Article  CAS  Google Scholar 

  9. Sullivan NL, Tzeranis DS, Wang Y, So PTC, Newman DK (2011) Quantifying the dynamics of bacterial secondary metabolites by spectral multiphoton microscopy. ACS Chem Biol 6:893–899

    Article  CAS  Google Scholar 

  10. San K-Y, Bennett GN, Berríos-Rivera SJ, Vadali RV, Yang Y-T, Horton E, Rudolph FB, Sariyar B, Blackwood K (2002) Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metab Eng 4:182–192

    Article  CAS  Google Scholar 

  11. Wos M, Pollard P (2006) Sensitive and meaningful measures of bacterial metabolic activity using NADH fluorescence. Water Res 40:2084–2092

    Article  CAS  Google Scholar 

  12. Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE, Boshoff HI (2011) Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis. PLoS Pathog 7:e1002287

    Article  CAS  Google Scholar 

  13. Dietrich LEP, Okegbe C, Price-Whelan A, Sakhtah H, Hunter RC, Newman DK (2013) Bacterial community morphogenesis is intimately linked to the intracellular redox state. J Bacteriol 195(7):1371–1380

    Article  CAS  Google Scholar 

  14. Denning GM, Iyer SS, Reszka KJ, O'Malley Y, Rasmussen GT, Britigan BE (2003) Phenazine-1-carboxylic acid, a secondary metabolite of Pseudomonas aeruginosa, alters expression of immunomodulatory proteins by human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 285:L584–L592

    Article  CAS  Google Scholar 

  15. Kito N, Ohnishi Y, Kagami M, Ohno A (1974) Reduction by a model of NAD(P)H. Construction of electron bridges. Chem Lett 3:353–356

    Google Scholar 

  16. Wang Y, Newman DK (2008) Redox reactions of phenazine antibiotics with ferric (hydr)oxides and molecular oxygen. Environ Sci Technol 42:2380–2386

    Article  CAS  Google Scholar 

  17. Burton RM, Kaplan NO (1963) The reaction of reduced pyridine nucleotides with acid. Arch Biochem Biophys 101:150–159

    Article  CAS  Google Scholar 

  18. Burton RM, Kaplan NO (1963) The reaction of diphosphopyridine nucleotide and related pyridinium salts with alkali. Arch Biochem Biophys 101:139–149

    Article  CAS  Google Scholar 

Download references

Acknowledgments

S.E.K. was supported by the National Science Foundation Graduate Research Fellowship Program, and D.K.N. is a Howard Hughes Medical Institute (HHMI) Investigator. We thank the HHMI for supporting our work. N. Glasser provided constructive criticism of earlier drafts of this chapter.

Author information

Authors and Affiliations

  1. Biology Department, Massachusetts Institute of Technology, Cambridge, MA, USA

    Suzanne E. Kern

  2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Suzanne E. Kern

  3. Division of Biology and Bioengineering, California Institute of Technology, Howard Hughes Medical Institute, Pasadena, CA, USA

    Alexa Price-Whelan

  4. Department of Biological Sciences, Columbia University, New York, NY, USA

    Alexa Price-Whelan

  5. Division of Biology and Bioengineering, Division of Geological and Planetary Sciences, Howard Hughes Medical Institute, California Institute of Technology, 1200 E. California Blvd. Mail Code 147-75, Pasadena, CA, 91125, USA

    Dianne K. Newman

Authors
  1. Suzanne E. Kern
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexa Price-Whelan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dianne K. Newman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dianne K. Newman .

Editor information

Editors and Affiliations

  1. MRC Centre for Molecular Bacteriology and Infection (CMBI) Department of Life Sciences, Imperial College, London, UK

    Alain Filloux

  2. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

    Juan-Luis Ramos

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kern, S.E., Price-Whelan, A., Newman, D.K. (2014). Extraction and Measurement of NAD(P)+ and NAD(P)H. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0473-0_26

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-0472-3

  • Online ISBN: 978-1-4939-0473-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation