Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes

Kenneth Olson, Yan Gao, Faihaan Arif, Kanika Arora, Shivali Patel, Eric Deleon, Karl D. Straub

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3=,6=-di(O-thiosalicyl)fluorescein, SSP4), and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n = 1–7) and H2S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

Original languageEnglish (US)
Pages (from-to)R340-R346
JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
Volume313
Issue number4
DOIs
StatePublished - Oct 1 2017

Keywords

  • Antioxidants
  • Fluorescent indicators
  • Reactive sulfide species
  • ROS

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

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