Depth-dependent flourescent quenching in micelles and membranes

Blatt, Edward; Sawyer, William H.

doi:10.1016/0304-4157(85)90003-6

Cited by 177 publications

(94 citation statements)

References 110 publications

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“…This approach was complemented with the second class of quenchers, spin-labeled fatty acids, which are lipid-based collisional quenchers. 5-DSA and 12-DSA act as molecular rulers, enabling assessment of the depth of location of the fluorophores with respect to the phospholipid bilayer (51) Taken together, we examined whether spatial proximity between positions 61 and 255 is retained upon interaction with a lipid surface by monitoring the signature spectral feature of pyrene. An apoE4 variant bearing Cys at these positions was constructed, which enabled us to selectively label with pyrene moiety employing the maleimide functional group.…”

Section: Effect Of Lipid Interaction Of Pyr-r61c/e255c/apoe4 On Pyrenmentioning

confidence: 99%

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

Drury¹,

Narayanaswami²

2005

Journal of Biological Chemistry

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Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wildtype apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/ E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another. Apolipoprotein E (apoE)1 belongs to the super family of exchangeable apolipoproteins that are major constituents of lipoproteins (1). It plays a crucial role in lipid homeostasis, not only in the plasma but also in the brain. ApoE exerts a protective effect against development of atherosclerosis (2, 3) by promoting cellular uptake (4) and clearance of triglyceride-rich lipoproteins from blood (5, 6). In addition, apoE in macrophages has a direct antiatherogenic effect independent of alterations in plasma lipoproteins (7) by promoting cholesterol efflux (8) from cell lining of arteries by a process termed reverse cholesterol transport (9 -11). The role of apoE in cholesterol homeostasis in the brain is emerging with evidence of its protective role against neuronal injury and neurodegeneration (12).In humans, apoE displays polymorphism with three major genetic variants identified in the population, apo⑀2, -⑀3, and -⑀4, with allelic frequencies of 8%, 77%, and 14%, respectively, determining six apoE phenotypes, apoE2/E2, apoE3/E3, apoE4/ E4, apoE2/E3, apoE2/E4, and apoE3/E4 (13). The apoE isoforms determine the atherogenic fate of the lipoproteins on which they reside. Although the apoE2 isoform is associated with type III hyperlipoproteinemia, peripheral atherosclerosis, and accumulation of remnant lipoproteins, apoE4 is consistent...

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Section: Effect Of Lipid Interaction Of Pyr-r61c/e255c/apoe4 On Pyrenmentioning

confidence: 99%

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

Drury¹,

Narayanaswami²

2005

Journal of Biological Chemistry

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“…This is also apparent in fig.1 which shows little difference in the lifetime quenching by 5-NS and 16-NS at both temperatures. a Partition coefficient determined by the method of Blatt and Sawyer [7] b Fluorescence lifetime in the absence of quencher (iv) At both temperatures the value of V is significantly less for 16-NS than for 5-NS.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescent and spin-labeled fatty acids have been used extensively to study the physical nature of microenvironments at a graded series of depths in the lipid bilayer [7,8]. Here, we examine the possibility of gross bending distortions of the acyl chains by measuring the quenching of n-(9-anthroyloxy) fatty acids by n-doxylstearates in situations where the fluorophore and quenching groups are at different transverse positions in the bilayer.…”

Section: Introductionmentioning

confidence: 99%

Vertical fluctuations of phospholipid acyl chains in bilayers

1987

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The possibility of vertical displacement of acyl chains in lipid bilayers has been examined by quenching the fluorescence of 2-(9-anthroyloxy)palmitic acid with 5-and 16-doxylstearates in dipalmitoylphosphatidylcholine unilamellar vesicles. Measurement of lifetime and steady-state quenching showed that the dynamic component of quenching was independent of the transverse position of the quencher indicating that a quencher at the 16-position could interact with a fluorophore at the 2-position with high frequency. The differences in steady-state quenching could be accounted for by the differences in the static component of quenching. The results provide further evidence for rapid vertical displacements of acyl chains in phospholipid bilayers.

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“…A teoria da supressão fluorescente tem sido descrita na literatura [140][141][142][143][144][145][146][147][148][149] e basicamente é governada pela Equação de Stern-Volmer (Equação 1):…”

Section: Determinação De Fármacos Baseada Na Supressão De Fluorescênciaunclassified

Aplicação e avanços da espectroscopia de luminescência em análises farmacêuticas

et al. 2008

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Recebido em 20/10/06; aceito em 30/11/07; publicado na web em 4/9/08 APPLICATION AND ADVANCES IN THE LUMINESCENCE SPECTROSCOPY IN PHARMACEUTICAL ANALYSES. This paper provides a review on the latest advances and applications of the luminescence spectroscopy for the development of pharmaceuticals analyses methods, basically based on the photo-and chemiluminescence. The different forms of the drugs determination on pharmaceuticals through the fluorescence and chemiluminescence are discussed. The analyses include the drugs native fluorescence (liquid and solid-phases); the fluorescence from the oxidizing or reducing forms of the drug; the fluorescence from the chemical derivatization and their photochemistry and hydrolysis reactions. The quenching of luminescence and chemiluminescence generation for the pharmaceutical quantification are also shown. Finally, the trends and future perspectives of the luminescence spectroscopy in the field of the pharmaceutical research are discussed.Keywords: luminescence spectroscopy; drugs; cosmetics analysis; introdução Devido ao progresso contínuo em técnicas e instrumentos, a indústria farmacêutica busca o emprego de metodologias que ofereçam maior sensibilidade, seletividade, robustez, precisão, exatidão e rapidez. Neste sentido, um grande número de equipamentos de cromatografia líquida de alta eficiência (CLAE) com diversos tipos de detecção domina este ramo industrial. Contudo, o advento da sofisticação de instrumentação automatizada de espectrômetros de luminescência poderá resultar em um substancial aumento de aplicações desta técnica instrumental na indústria farmacêutica, principalmente, no que se refere à análise diretamente na matriz do medicamento.A espectroscopia de luminescência é uma ferramenta analítica extremamente sensível e tem sido amplamente aplicada na resolução de problemas que requerem baixos limites de detecção, podendo ser facilmente encontrados na literatura muitos trabalhos mostrando análises ao nível de traços.1-6 A sensibilidade inerente a esta técnica é consideravelmente maior em comparação a outras metodologias, entre elas a espectrofotometria UV/visível, apresentando limites de detecção de até três ordens de grandeza menores, os quais normalmente se encontram na faixa de ng mL -1 , sendo conseqüência do baixo sinal de fundo que as medidas fluorescentes apresentam. Diferentemente do que ocorre no fenômeno da luminescência, nas medidas de absorbância o sinal está associado à atenuação da intensidade de um feixe de radiação por uma espécie absorvente. Nos casos onde a quantidade da espécie é pequena, esta atenuação é menor, tornando assim a detecção da espécie difícil. Outra característica importante da luminescência diz respeito à seletividade, porque espécies com rendimentos quânticos de fluorescência apreciáveis são menos comuns do que substâncias cuja fluorescência não pode ser detectada. Desta forma, moléculas fluorescentes apresentam comprimento de onda característico de excitação e/ou emissão. Adicionalmente, o comprimento de onda de excitação d...

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Depth-dependent flourescent quenching in micelles and membranes

Cited by 177 publications

References 110 publications

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

Vertical fluctuations of phospholipid acyl chains in bilayers

Aplicação e avanços da espectroscopia de luminescência em análises farmacêuticas

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