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

2 Citations (Scopus)

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

Fingerprint

Porphyrins
Fluorescence
Metals
Catalase
Enzymes
Fluorescein
Albumins
Reactive Oxygen Species
Diamide
Iodoacetates
Hemeproteins
Light
Sodium Azide
Hemin
Dithiothreitol
Fluorescence Spectrometry
Sulfides
Fluorescence Microscopy
Chlorides
Hemoglobins

Keywords

  • Antioxidants
  • Fluorescent indicators
  • Reactive sulfide species
  • ROS

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes. / Olson, Kenneth; Gao, Yan; Arif, Faihaan; Arora, Kanika; Patel, Shivali; Deleon, Eric; Straub, Karl D.

In: American Journal of Physiology - Regulatory Integrative and Comparative Physiology, Vol. 313, No. 4, 01.10.2017, p. R340-R346.

Research output: Contribution to journalArticle

Olson, Kenneth ; Gao, Yan ; Arif, Faihaan ; Arora, Kanika ; Patel, Shivali ; Deleon, Eric ; Straub, Karl D. / Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes. In: American Journal of Physiology - Regulatory Integrative and Comparative Physiology. 2017 ; Vol. 313, No. 4. pp. R340-R346.
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AU - Olson, Kenneth

AU - Gao, Yan

AU - Arif, Faihaan

AU - Arora, Kanika

AU - Patel, Shivali

AU - Deleon, Eric

AU - Straub, Karl D.

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AB - 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.

KW - Antioxidants

KW - Fluorescent indicators

KW - Reactive sulfide species

KW - ROS

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