Intramolecular quenching of excited singlet states by stable nitroxyl radicals

Green, S. A.; Simpson, Daniel J.; Zhou, Guijiang; Ho, Pauline; Blough, Neil V.

doi:10.1021/ja00176a038

Cited by 253 publications

(224 citation statements)

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“…The nitroxide labels used have a weak absorption in the UV, 430 nm ϭ 14 M Ϫ1 cm Ϫ1 (29). From the absorption tail a value of E ϭ 100 M Ϫ1 at the pyrene excitation wavelength ( exc ϭ 345 nm) is determined.…”

Section: Methodsmentioning

confidence: 99%

Topography of Nicotinic Acetylcholine Receptor Membrane-embedded Domains

Barrantes¹,

Antollini²,

Blanton³

et al. 2000

Journal of Biological Chemistry

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The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in ␣M1, ␣M4, ␥M1, and ␥M4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear ␣-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.The muscle and electric organ nicotinic acetylcholine receptor (AChR) 1 is a pentameric integral transmembrane protein of homologous ␣ 2 ␤␥␦ subunits. The AChR belongs to a superfamily of ligand-gated ion channels, together with the glycine receptor, a subtype of the serotonin receptor (5-HT 3 ), and the GABA A receptor (1-4). Each AChR subunit contains a relatively large amino-terminal extracellular domain of ϳ200 amino acids followed by four hydrophobic domains of 20 -30 amino acids in length (M1-M4) connected by hydrophilic loops of varying length and ending with a very short extracellular carboxyl terminus (reviewed in Ref. 5).Although the exact topology of the AChR relative to the membrane has not yet been determined unambiguously, it is usually accepted that the four hydrophobic segments M1-M4 correspond to transmembrane (TM) domains (6 -7). There is still contradictory evidence on their secondary structure. The original postulation of a four-helix bundle with an all-helical secondary structure (8) has been challenged by the results of cryoelectron microscopy of frozen AChR tubules (9 -10) and computer-aided molecular modeling indicating that the dimensions of the AChR TM region are not compatible with a pentameric four-helix bundle (11). Site-directed mutagenesis data combined with patch clamp electrophysiology, and results from photoaffinity labeling with noncompetitive channel blockers, support the notion that the M2 domain lines the walls of the ion channel proper and are indicative of ␣-helical periodicity in the residues exposed ...

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Section: Methodsmentioning

confidence: 99%

Topography of Nicotinic Acetylcholine Receptor Membrane-embedded Domains

Barrantes¹,

Antollini²,

Blanton³

et al. 2000

Journal of Biological Chemistry

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“…Blough and co-workers have shown that these covalently-linked nitroxidefluorophore adducts can be employed as very sensitive optical sensors of radical/redox reactions (8)(9)(10)(11). Scaiano and co-workers have also developed a number of prefluorescent probes allowing radical and/or antioxidant detection in homogeneous and heterogeneous systems (12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Study on the interaction between nitroxide free radical and conjugated polyelectrolytes by fluorimetry

Dou

2008

Luminescence

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In this work, we studied the fluorescence quenching of the anionic conjugated polyelectrolyte PPE-SO 3 by the paramagnetic species 2,2,6,6-tetramethylpiperidine-N-oxide free radical (TEMPO) in aqueous solution. At low quencher concentration the Stern-Volmer constant is 94 mol/L; as the quencher concentration increases the Stern-Volmer plots become superlinear. Ascorbic acid is used to reduce TEMPO to the corresponding hydroxylamine and the PPE-SO 3 fluorescence is fully recovered. Under a large excess of ascorbic acid over TEMPO, the rise of fluorescence followed pseudo-first-order kinetics. IntroductionConjugated polyelectrolytes (CPEs) are π-conjugated polymers that feature ionic solubilizing side groups (1). These materials show many of the interesting and useful properties exhibited by π-conjugated polymers, such as high fluorescence quantum yields, unique solution behaviour, the ability to interact electrostatically with other charged species, and an extraordinarily high sensitivity to fluorescence quenchers (2). This latter property has been exploited in sensing experiments, in which a variety of organic and inorganic analytes and biomolecules can be detected at picomolar concentration levels (3-6).Nitroxides are persistent free radicals frequently used as radical scavengers or as quenchers of excited states (7). Blough and co-workers have shown that these covalently-linked nitroxidefluorophore adducts can be employed as very sensitive optical sensors of radical/redox reactions (8-11). Scaiano and co-workers have also developed a number of prefluorescent probes allowing radical and/or antioxidant detection in homogeneous and heterogeneous systems (12)(13)(14).However, there is no report of studies on the interaction between nitroxides radicals and conjugated polyelectrolytes until now and the mechanism is also not clear. In an effort to construct highly sensitive optical probes of radical/redox species, we sought to exploit both the radical trapping and fluorescence quenching properties of the nitroxides by constructing a system that contains nitroxides and conjugated polyelectrolytes.In this work, we studied the fluorescence quenching of the sulphonate-substituted poly(phenylene ethynylene) PPE-SO 3 by the paramagnetic species 2,2,6,6-tetramethylpiperidine-N-oxide free radical (TEMPO) in aqueous solution (structures in Fig. 1). PPE-SO 3 is a water-and alcohol-soluble polymer that exhibits a strong blue or green fluorescence (15, 16). The possible mechanism of fluorescence quenching was studied. Additionally, chemical reduction of the TEMPO to a diamagnetic hydroxylamine can lead a fluorescence recovery of PPE-SO 3 . ExperimentalChemicals PPE-SO 3 was synthesized according to a previously described method (16), and the structure (Fig. 1)

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“…Covalently linking a nitroxide moiety to a fluorescent structure possessing excitation and emission profiles of biological relevance (Morrow et al, 2010), e.g. rhodamine, efficiently quenches the excited state that leads to fluorescence (Ahn et al, 2012;Blough and Simpson, 1988;Green et al, 1990). The removal of the nitroxide free radical through a one-electron reduction to the non-radical hydroxylamine, removes this quenching effect resulting in the restoration of the typical fluorescence of the fluorophore (Blough and Simpson, 1988).…”

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confidence: 99%