UTILIZATION OF HEAVY-ATOM EFFECT QUENCHING OF PYRENE FLUORESCENCE TO DETERMINE THE INTRAMEMBRANE DISTRIBUTION OF HALOTHANE (original) (raw)
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The Journal of Physical Chemistry, 1993
The behavior of N-(1-pyrenesulfonyl)dipalmitoyl-L-a-phosphatidylethanolamine (pyrene-DPPE) embedded in dimyristoyl-L-a-phosphatidylcholine (DMPC) monolayers at the gas-water interface has been examined by surface pressurearea isotherm measurements and steady-state and time-resolved fluorescence spectroscopy. The pyrene moiety of pyreneDPPE markedly alters the packing characteristics from those of the pure lipid DPPE, contributing significantly to the area per molecule. Steady-state fluorescence spectra showed monomer emission only, and a nonlinear increase in fluorescence intensity with concentration of pyreneDPPE was observed, which can be attributed to oxygen quenching. Time-resolved fluorescence measurements yielded single-exponential decays for the pyrene chromophore, providing evidence that pyrene-DPPE was not aggregated in the monolayer film. The lifetime of pyrene-DPPE was also found to increase with monolayer compression. The presence of iodide ions in the subphase efficiently quenched the fluorescence of pyreneDPPE in the monolayer. The degree of quenching was found to be independent of the molecular packing density, suggesting that the pyrene chromophore is located in the headgroup region of the monolayer. Measurements of the steady-state fluorescence intensity as a function of area per molecule showed pyrene-DPPE to be strongly susceptible to oxygen quenching. The degree of oxygen quenching was found to decrease with compression, accounting for the increase in the fluorescence lifetime of the excited pyrene with increasing surface pressure.
Biophysical Journal, 2001
Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2 H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1 H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long-and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.
Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes
Biophysical Chemistry, 1985
The effect of Ca*+ on the molecular mobility in dipalmitoylphosphatidylcholine membranes was studied hy steady-state and time-resolved measurements of fluorescence anisotropy. The fluorescence anisotropy decay of 16diphenyl-1.3,Shexatriene in the hydrocarbon region indicated that the free volume of molecular rotation became more restricted when the CL?+ concentration was increased. The decrease of the molecular mobility was observed from 1 mM Ca*+, at which the number of bound CaZf is much less than that of the total lipid molecules. A distinct difference between Ca2+ and Mg'+ effects suggested that the change in various membrane properties was induced by the binding of these ions. From these results we propose a long-range attractive interaction between bound Ca" and the polar head groups of distant phosphatidylcholine molecules.
Fluorescence quenching of gramicidin D in model membranes by halothane
Canadian Journal of Chemistry, 2007
Inhaled anesthetics were introduced in surgery over a century ago. To this day, the molecular mechanism of anesthetic action remains largely unknown. However, ion-channels of neuronal membranes are believed to be the most- likely molecular targets of inhaled anesthetics. In the study presented here, we investigated the interaction of a simplified ion-channel system, gramicidin, with halothane, a small haloalkane inhaled anesthetic in various environments. Fluorescence-quenching experiments of gramicidin D in dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles (LUVS) have shown that halothane can directly interact with the ion channel (KSV = 66 M–1). Halothane quenched the fluorescence from tryptophan residues located at the lipid bilayer – aqueous interfaces as well as those tryptophans located deeper in the bilayer. Quenching data from gramicidin D in sodium dodecyl sulfide (SDS) micelles revealed that the tryptophan residues located at the micelle–solvent interface were ...
Chemistry and Physics of Lipids, 1984
The steady state fluorescence anisotropy (r s) of l-acyl-2-cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model-and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased r s for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the rs-value for DPH strongly, whereas the rs-value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the r s of DPH was higher than that of PnPC. No large differences between the rs-values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-paimitoyl-2-arachidonyl PC.
Use of the fluorescent dye, Pyrene, to study the dynamic aspects of membrane structure
1974
Synopsis. Lateral diffusion of the fluorescent molecule, Pyrene, in phospholipid vesicles and mitochondrial membranes is demonstrated. Pyrene forms excited state dimers (excimers) in a diffusion limited reaction which depends upon membrane composition, temperature and pyrene concentration. The results obtained show that the classical theory of diffusion are not exactly applicable. The emission spectrum of the pyrene monomer in the membrane phase is highly structured and at 77 K a Shpol'skii spectrum is obtained (Shpol'skii, 196o). The formation of excimers in membranes affords evidence for the fluid mosaic model for membrane structure.
Changes of the Membrane Lipid Organization Characterized by Means of a New Cholesterol-Pyrene Probe
Biophysical Journal, 2007
We synthesized 3b-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe selfassociation processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K DOPC) K POPC) K DMPC. K DMPC/15 mol % Chol. K DMPC/30 mol % Chol. This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I 1-I 5) and excimer (I E) emission bands. In model membranes, I 1 /I 3 and I E /I 3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse d-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse d-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.
Journal of Photochemistry and Photobiology A: Chemistry, 1995
Probe location in biological membranes is important and predominant positions of five pyrene derivatives in erythrocyte membranes are inferred from their fluorescence properties. The distribution of the probes along the normal to the bilayer surface was studied by fluorescence quenching; the quenching etlicieney was correlated with the nature and the rate of access of the quencher to the solubilisation site of the probe. Probes and iodide quenchers with known specificity for certain membrane areas were used to survey membrane dynamics. These probes included pyrene butyric acid (PBA) which partly partitions in water, pyrene carboxy aldehyde (PCA) which binds primary on the membrane surface and pyrene (PY), benzo(a)pyrene (BaP) and pyrene decanoie acid (PDA), which insert at different depths in the hydrocarbon core of membranes. The set of quenchers includes hydrophilic iodobenzene, amphiphilic iodobenzoie and l-iodo propanoie acids and the long-chain alkyi iododecanoic acid. Comparison of steady state and time-resolved quenching experiments indicates that dynamic quenching is predominant since it contributes by more than 75% to the total quenching. These data suggest that PY, PDA and BaP intercalate in different zones in the bilaver. PY is probably diffusing in a relatively large area of the lipid matrix between the centers of the lipid layers, PDA and BaP residing preferentially in a restricted central region of the erythroeyte membrane.
Fluorescence studies of lipid regular distribution in membranes
Chemistry and Physics of Lipids, 2002
This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of pyrene-labeled PC. The original and the extended hexagonal superlattice model are discussed. The second part focuses on the fluorescence studies of sterol regular distributions in membranes. The experimental evidence for sterol superlattice formation obtained from the fluorescent sterol (i.e. dehydroergosterol) and non-sterol fluorescent probes (e.g. DPH and Laurdan) are evaluated. Prospects and concerns are given with regard to the sterol regular distribution. The third part deals briefly with the evidence for polar headgroup superlattices. The emphasis of this article is placed on the new concept that membrane properties and activities, including the activities of surface acting enzymes, drug partitioning, and membrane free volume, are fine-tuned by minute changes in the concentration of bulky lipids (e.g. sterols and pyrene-containing acyl chains) in the vicinities of the critical mole fractions for superlattice formation.
Halothane Changes the Domain Structure of a Binary Lipid Membrane
2012
X-ray and neutron diffraction studies of a binary lipid membrane demonstrate that halothane at physiological concentrations produces a pronounced redistribution of lipids between domains of different lipid types identified by different lamellar d-spacings and isotope composition. In contrast, dichlorohexafluorocyclobutane (F6), a halogenated nonanesthetic, does not produce such significant effects. These findings demonstrate a specific effect of inhalational anesthetics on mixing phase equilibria of a lipid mixture.