Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red

Free Radic Biol Med. 2011 Jul 1;51(1):153-9. doi: 10.1016/j.freeradbiomed.2011.03.016. Epub 2011 Mar 17.

Abstract

The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF(•) ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/(•)OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF(•) ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH₂). Both RSF(•) ‾ and RSFH₂, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Benzoquinones / chemical synthesis
  • Cyclic N-Oxides / metabolism
  • Fluorescent Dyes
  • Hydrogen Peroxide / chemical synthesis
  • Light
  • NAD / chemistry
  • NAD / metabolism
  • Oxazines / chemical synthesis
  • Oxazines / chemistry*
  • Oxidation-Reduction
  • Oxidative Stress*
  • Photosensitizing Agents / chemistry*
  • Reactive Oxygen Species / chemical synthesis*
  • Staining and Labeling
  • Superoxide Dismutase / metabolism

Substances

  • Benzoquinones
  • Cyclic N-Oxides
  • Fluorescent Dyes
  • Oxazines
  • Photosensitizing Agents
  • Reactive Oxygen Species
  • dihydroresorufin
  • NAD
  • Amplex Red
  • semiquinone radicals
  • resorufin
  • 5,5-dimethyl-1-pyrroline-1-oxide
  • Hydrogen Peroxide
  • Superoxide Dismutase