Oxyradical-induced GFP damage and loss of fluorescence

Abeer A. Alnuami, Buthaina Zeedi, Shahnaz M. Qadri, S. Salman Ashraf

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

Abstract

Small amounts of highly reactive oxygen species (oxyradicals) are normal by-products of cellular metabolism. However, under certain conditions large amounts of oxyradicals are generated inside cells which may cause extensive cellular damage. Not surprisingly, a large number of disease states have been linked to oxidative stress, including cancer, diabetes, Parkinson's disease, Alzheimer's disease, and heart disease. Previously, we had shown that fluorescence spectroscopy could be used to study the pH-dependence of GFP denaturation with various agents. In this report, we show that GFP readily loses its auto-fluorescence upon exposure to oxyradicals as measured by fluorescence spectroscopy. We further show that oxyradical scavengers can prevent this loss of GFP fluorescence, thus oxyradical-induced loss of GFP fluorescence could be used to screen for antioxidants. We have evaluated various parameters which could affect the sensitivity of this GFP-based oxyradical scavenging assay, such as concentration H2O2 used to produce oxyradicals, pH of the buffer, as well as UV intensity. Surprisingly we found that pH had a very dramatic effect on oxyradical-induced GFP damage. GFP was found to be most susceptible to oxyradical-induced damage at pH 6.5, and least susceptible at pH 8.5. This is the first demonstration that GFP loses its fluorescence upon exposure to oxyradicals. Furthermore, the data presented here suggest that GFP could be used to develop assays to screen for antioxidants or radical scavengers.

Original languageEnglish
Pages (from-to)182-186
Number of pages5
JournalInternational Journal of Biological Macromolecules
Volume43
Issue number2
DOIs
Publication statusPublished - Aug 15 2008

Keywords

  • Green fluorescent protein
  • Oxidative stress
  • Oxyradical

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Economics and Econometrics
  • Energy(all)

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