Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes

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(-thiosalicyl)fluorescein, SSP4), and HS (7-azido-4-methylcoumarin, AzMC) previously activated by HO, a mixed polysulfide (HS, = 1-7) and HS, 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.

Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

“…Heme quenching of pocket tryptophan and tyrosine residue fluorescence is known to limit the use of tryptophan or tyrosine fluorescence quenching assays to study ligand cobinding to heme-bound proteins (14). An additional study warned against the use of fluorescence-based substrates in assays studying heme-binding proteins (e.g., catalase, hemoglobin) because heme quenching of the fluorescent signal causes assay artifacts (15). Consistent with these studies, we found that heme dose-dependently quenched the fluorescence of fluorophores with excitation and emission wavelengths ranging from 495 nm to 785 nm ( Fig.…”

“…To support our ITC results, we performed solution NMR spectroscopy-a structural technique that provides information about the dynamics and structure (via changes to the chemical environment of individual atoms) in a protein at atomic resolution. Titration of NCoR ID peptide into 15 N-labeled REV-ERBβ LBD can 1) validate that the interaction occurs in the presence of heme, 2) qualitatively reflect the strength of the interaction relative to apo (ligand-free)-REV-ERBβ LBD, and 3) highlight the residues most likely involved in binding (16). We previously obtained backbone NMR chemical shift assignments for REV-ERBβ LBD (17).…”

“…To generate heme-bound protein, 1.25 molar equivalents of heme prepared in 0.2 M NaOH as a 50 mM stock were added to 15 N-labeled REV-ERBβ LBD (200 μM) or 2 H, 15 N, 13 C-labeled REV-ERBβ LBD (1 mM) in HEPES buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 0.5 mM EDTA). TROSY-based 3D HNCO, HNCA, HN(CA)CB, HN(COCA)CB, HN(-CO)CA, and HN(CA)CO experiments were collected at 298 K using the heme-bound 2 H, 15 N, 13 C-REV-ERBβ LBD. 2D [ 1 H, 15 N]-TROSY-HSQC data were collected for heme-bound 15 N-labeled REV-ERBβ LBD at 298 K with addition of NCoR ID1 or ID2 peptides prepared as 50 mM stock in DMSO and analyzed in NMRViewJ.…”

“…(B) Thermodynamic parameters calculated from global fitting analysis of replicate (N ≥ 2) ITC experiments (mean with 95% confidence intervals calculated for each parameter as reported inTable S1). (C) Representative peaks from 2D [ 1 H,15 N]-TROSY-HSQC spectra of 15 N-labeled REV-ERBβ LBD with or without heme titrated with NCoR ID1 or ID2 peptide. (D) 2D [ 1 H,15 N]-TROSY-HSQC spectral overlays of heme-bound REV-ERBβ LBD in the presence of vehicle (0.8% DMSO; black peaks) or 2 molar equivalents of ID1 or ID2 peptide (red peaks).…”

“…(C) Representative peaks from 2D [ 1 H,15 N]-TROSY-HSQC spectra of 15 N-labeled REV-ERBβ LBD with or without heme titrated with NCoR ID1 or ID2 peptide. (D) 2D [ 1 H,15 N]-TROSY-HSQC spectral overlays of heme-bound REV-ERBβ LBD in the presence of vehicle (0.8% DMSO; black peaks) or 2 molar equivalents of ID1 or ID2 peptide (red peaks). Residues with the largest CSP changes or line broadening were mapped as red or black spheres, respectively, onto the crystal structure of heme-bound REV-ERBβ LBD (PDB 3CQV).…”