Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Molecular imaging of hydrogen peroxide produced for cell signaling

Nature Chemical Biology volume 3, pages 263–267 (2007)Cite this article

A Corrigendum to this article was published on 01 June 2007

This article has been updated

Abstract

Hydrogen peroxide (H2O2) is emerging as a newly recognized messenger in cellular signal transduction1,2. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems with molecular specificity. Here we report the synthesis and application of Peroxy Green 1 (PG1) and Peroxy Crimson 1 (PC1), two new fluorescent probes that show high selectivity for H2O2 and are capable of visualizing endogenous H2O2 produced in living cells by growth factor stimulation, including the first direct imaging of peroxide produced for brain cell signaling. The combined features of reactive oxygen species selectivity, sensitivity to signaling levels of H2O2, and live-cell compatibility presage many new opportunities for PG1, PC1 and related synthetic reagents for exploring the physiological roles of H2O2 in living systems with molecular imaging.

*Note: In the version of this article initially published, the second author's last name is misspelled. The author's name should read Orapim Tulyathan. The error has been corrected in the HTML and PDF versions of the article.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectroscopic responses and selectivities for H2O2 probes.
Figure 2: Live-cell H2O2 imaging with PG1 and PC1.
Figure 3: Molecular imaging of H2O2 produced by growth factor signaling.
Figure 4: 4 H2O2 and growth factor signaling in living neurons.

Similar content being viewed by others

Change history

  • 03 May 2007

    changed from Tulyanthan to Tulyathan, first n removed

References

  1. Rhee, S.G. H2O2, a necessary evil for cell signaling. Science 312, 1882–1883 (2006).

    Article  Google Scholar 

  2. Finkel, T. Oxidant signals and oxidative stress. Curr. Opin. Cell Biol. 15, 247–254 (2003).

    Article  CAS  Google Scholar 

  3. Beckman, K.B. & Ames, B.N. The free radical theory of aging matures. Physiol. Rev. 78, 547–581 (1998).

    Article  CAS  Google Scholar 

  4. Stone, J.R. & Yang, S. Hydrogen peroxide: a signaling messenger. Antioxid. Redox Signal. 8, 243–270 (2006).

    Article  CAS  Google Scholar 

  5. Sundaresan, M., Yu, Z.-X., Ferrans, V.J., Irani, K. & Finkel, T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296–299 (1995).

    Article  CAS  Google Scholar 

  6. Bae, Y.S. et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272, 217–221 (1997).

    Article  CAS  Google Scholar 

  7. Ohba, M., Shibanuma, M., Kuroki, T. & Nose, K. Production of hydrogen peroxide by transforming growth factor-β1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J. Cell Biol. 126, 1079–1088 (1994).

    Article  CAS  Google Scholar 

  8. Kimura, T., Okajima, F., Sho, K., Kobayashi, I. & Kondo, Y. Thyrotropin-induced hydrogen peroxide production in FRTL-5 thyroid cells is mediated not by adenosine 3′,5′-monophosphate, but by Ca2+ signaling followed by phospholipase-A2 activation and potentiated by an adenosine derivative. Endocrinology 136, 116–123 (1995).

    Article  CAS  Google Scholar 

  9. Mukhin, Y.V. et al. 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts. Biochem. J. 347, 61–67 (2000).

    Article  CAS  Google Scholar 

  10. Lee, S.R., Kwon, K.S., Kim, S.R. & Rhee, S.G. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J. Biol. Chem. 273, 15366–15372 (1998).

    Article  CAS  Google Scholar 

  11. Salmeen, A. et al. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate. Nature 423, 769–773 (2003).

    Article  CAS  Google Scholar 

  12. Guyton, K.Z., Liu, Y., Gorospe, M., Xu, Q. & Holbrook, N.J. Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J. Biol. Chem. 271, 4138–4142 (1996).

    Article  CAS  Google Scholar 

  13. Schmidt, K.N., Amstad, P., Cerutti, P. & Baeuerle, P.A. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-κB. Chem. Biol. 2, 13–22 (1995).

    Article  CAS  Google Scholar 

  14. Avshalumov, M.V. & Rice, M.E. Activation of ATP-sensitive K+ (K(ATP)) channels by H2O2 underlies glutamate-dependent inhibition of striatal dopamine release. Proc. Natl. Acad. Sci. USA 100, 11729–11734 (2003).

    Article  CAS  Google Scholar 

  15. Wood, Z.A., Poole, L.B. & Karplus, P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300, 650–653 (2003).

    Article  CAS  Google Scholar 

  16. Woo, H.A. et al. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation. Science 300, 653–656 (2003).

    Article  CAS  Google Scholar 

  17. Biteau, B., Labarre, J. & Toledano, M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 425, 980–984 (2003).

    Article  CAS  Google Scholar 

  18. Lee, J.-W. & Helmann, J.D. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 440, 363–367 (2006).

    Article  CAS  Google Scholar 

  19. Stadtman, E.R., Van Remmen, H., Richardson, A., Wehr, N.B. & Levine, R.L. Methionine oxidation and aging. Biochim. Biophys. Acta 1703, 135–140 (2005).

    Article  CAS  Google Scholar 

  20. Soh, N. Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal. Bioanal. Chem. 386, 532–543 (2006).

    Article  CAS  Google Scholar 

  21. Hempel, S.L., Buettner, G.R., O'Malley, Y.Q., Wessels, D.A. & Flaherty, D.M. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146–159 (1999).

    Article  CAS  Google Scholar 

  22. Zhou, M., Diwu, Z., Panchuk-Voloshina, N. & Haugland, R.P. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162–168 (1997).

    Article  CAS  Google Scholar 

  23. Maeda, H. et al. Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism. Angew. Chem. Int. Ed. 43, 2389–2391 (2004).

    Article  CAS  Google Scholar 

  24. Belousov, V.V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).

    Article  CAS  Google Scholar 

  25. Chang, M.C., Pralle, A., Isacoff, E.Y. & Chang, C.J. A selective, cell-permeable optical probe for hydrogen peroxide in living cells. J. Am. Chem. Soc. 126, 15392–15393 (2004).

    Article  CAS  Google Scholar 

  26. Miller, E.W., Albers, A.E., Pralle, A., Isacoff, E.Y. & Chang, C.J. Boronate-based fluorescent probes for imaging cellular hydrogen peroxide. J. Am. Chem. Soc. 127, 16652–16659 (2005).

    Article  CAS  Google Scholar 

  27. Setsukinai, K.-I., Urano, Y., Kakinuma, K., Majima, H.J. & Nagano, T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 278, 3170–3175 (2003).

    Article  CAS  Google Scholar 

  28. Ullrich, A. et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418–425 (1984).

    Article  CAS  Google Scholar 

  29. Wong, R.W.C. & Guillaud, L. The role of epidermal growth factor and its receptors in mammalian CNS. Cytokine Growth Factor Rev. 15, 147–156 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the University of California, Berkeley, the Dreyfus Foundation, the Beckman Foundation, the American Federation for Aging Research, the Packard Foundation, and the US National Institute of General Medical Sciences (NIH GM 79465) for funding this work. E.W.M. thanks the Chemical Biology Graduate Program sponsored by the US National Institutes of Health (T32 GM066698) and a Stauffer fellowship for support. We thank D. Fortin and S. Szobota for providing neuronal cultures, and we thank K. McNeill (University of Minnesota) for the polymer-supported Rose Bengal. Confocal fluorescence images were acquired at the Molecular Imaging Center at UC Berkeley. We also thank A. Fischer at the UC Berkeley Tissue Culture Facility for expert technical assistance.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, 532A Latimer Hall, Berkeley, 94720, California, USA

    Evan W Miller & Christopher J Chang

  2. Department of Molecular and Cell Biology, University of California, Valley Life Sciences Addition, Berkeley, 94720, California, USA

    Orapim Tulyathan & Ehud Y Isacoff

Authors
  1. Evan W Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Orapim Tulyathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ehud Y Isacoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher J Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.W.M. performed all of the synthetic and imaging experiments. O.T. prepared the neuronal cultures and helped with the immunostaining experiments. C.J.C. and E.W.M. designed experimental strategies with help from E.Y.I. C.J.C. and E.W.M. wrote the paper.

Corresponding author

Correspondence to Christopher J Chang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Spectroscopic responses and selectivities for PG1 and PC1 in different buffers. (PDF 107 kb)

Supplementary Fig. 2

Relative responses of PG1, PC1 or DCFH to H2O2 or high-valent iron-oxo centers generated from heme and H2O2. (PDF 239 kb)

Supplementary Methods (PDF 115 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Miller, E., Tulyathan, O., Isacoff, E. et al. Molecular imaging of hydrogen peroxide produced for cell signaling. Nat Chem Biol 3, 263–267 (2007). https://doi.org/10.1038/nchembio871

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio871

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing