Fatty Acid Probes Research Articles

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S-acylation

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC ‘hole’ mutants and ‘bumped’ chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical–genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

The Road to Quantitative Lipid Biochemistry in Living Cells.

Traditional cell biological techniques are not readily suitable for studying lipid signaling events because genetic perturbations are much slower than the interconversion of lipids in complex metabolic networks. For this reason, novel chemical biological approaches have been developed. One approach is to chemically modify a lipid with a so-called "caging group" that renders it inactive, but this cage can be removed photochemically inside cells to release the bioactive molecule. These caged compounds offer unique advantages for studying the kinetics of cellular biochemistry and have been extensively used in the past. However, a limitation of conventional caged compounds is their ability to diffuse freely inside the cell, which does not permit localized activation below optical precision. This poses a challenge for studying lipid signaling as lipid function inside cells is tightly linked to their parent membrane. An ideal lipid probe should, therefore, be restricted to a single organelle membrane or preferentially to a single leaflet. We first demonstrated the plasma-membrane-specific photorelease of fatty acids by employing sulfonated caging groups. Using these caged fatty acid probes we demonstrated that lipid localization determines signaling outcome. Generalizing this approach, we designed a so-called "click cage" that can be coupled to lipids and offers the possibility to attach organelle targeting groups via click chemistry. Using this strategy, we have synthesized plasma membrane, lysosomal, mitochondria, and endoplasmic-reticulum-targeted lipids that can be used to dissect organelle-specific signaling events. To reduce the synthetic effort associated with generating caged compounds, we designed a coumarin triflate reagent that allows the direct functionalization of phosphate- or carboxylate-containing compounds. With this novel reagent, we synthesized a small library of photocaged G-protein-coupled receptor (GPCR) ligands to study the underlying lipid signaling dynamics. Most recently, we have focused on quantifying the kinetics of lipid signaling for different diacylglycerol (DAG) species using plasma-membrane-targeted caged DAGs. Using this approach, we quantitatively measured lipid-protein affinities and lipid transbilayer dynamics in living cells. After analyzing DAGs with different acyl chain length and saturation degree, we discovered that affinities can vary by up to an order of magnitude. This finding clearly shows that cells are able to distinguish between individual DAG species, thereby demonstrating that lipid diversity matters in cellular signal processing. Although the recent advances have yielded valuable tools to study lipid signaling, challenges remain on specifically targeting the different leaflets of organelle membranes. Furthermore, it is necessary to simplify the experimental approaches required for parametrizing and corroborating quantitative kinetic models of lipid signaling. In the future, we envision that the application of leaflet-specific caged lipids to model membrane systems will be of crucial importance for understanding lipid asymmetry.

Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate (NADPH), however, their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells. A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electron-donating system. Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide (NCDH) and created non-natural cofactor-dependent P450 catalysis. Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over 15,000 M−1 s−1 for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate. Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance. Upon fusing with an NCD-dependent formate dehydrogenase, fused proteins functioned as NCDH-specific P450 catalysts by using formate as the electron donor. Importantly, these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid. As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths, our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

Metabolic distribution of fatty acid to organelles is an essential biological process for energy homeostasis as well as for the maintenance of membrane integrity, and the metabolic pathways are strictly regulated in response to environmental stimuli. Herein, we report a fluorescent fatty acid probe, which bears an azapyrene dye that changes its absorption and emission features depending on the microenvironment polarity of the organelle into which it is transported. Owing to the environmental sensitivity of this dye, the distribution of the metabolically incorporated probe in non-polar lipid droplets, medium-polarity membranes, and the polar aqueous regions, can be visualized in different colors. Based on density scatter plots of the fluorophore, we demonstrate that the degradation of triacylglycerols in lipid droplets occurs predominantly via lipolysis rather than lipophagy in nutrition-starved hepatocytes. This tool can thus be expected to significantly advance our understanding of the lipid metabolism in living organisms.

PGC1α-mediated fatty acid oxidation promotes TGFβ1-induced epithelial-mesenchymal transition and metastasis of nasopharyngeal carcinoma

AimCancer cells frequently undergo metabolic reprogramming, which contributes to tumorigenicity and malignancy. Unlike primary cancers, during the process of invasion and distal dissemination, cancer cells are deficient in ATP due to damaged glucose transport. Cells need to rewire metabolic programs to overcome nutrient and energy crises, maintaining survival and forming metastasis. However, the underlying mechanism has not been well understood. We elucidated the metabolic alteration in TGFβ1-induced epithelial-mesenchymal transition (EMT) and metastasis of nasopharyngeal carcinoma (NPC). Main methodsFluorescent Bodipy fatty acid probe, UPLC-MS/MS analysis, β-oxidation assay, cellular ATP and NADPH/NADP measurement, and Oil Red-O staining were performed to evaluate the activation of FAO pathways in the TGFβ1-induced EMT of NPC cells. Three-dimensional (3D) invasion assay and metastatic animal model were applied to assess the invasive and metastatic capacity of NPC cells. Key findingsOur current findings reveal that PGC1α-mediated FAO promotes TGFβ1-induced EMT and metastasis of NPC cells. Mechanically, TGFβ1 up-regulates AMPKα1 to activate PGC1α, which transcriptionally boosts FAO-associated genes. The metabolic rewiring mediated by PGC1α facilitates EMT, invasion, and metastasis of NPC. SignificanceThe present study aims to establish the mechanistic connection between energy metabolic reprogramming and the aggressive phenotype of NPC. These actions further provide new opportunities for developing of novel therapeutics for NPC by targeting PGC1α/ FAO signaling.

Glycation Alters the Fatty Acid Binding Capacity of Human Serum Albumin.

Glycation significantly alters the physicochemical and biofunctional properties of proteins in foods and in vivo. In the present study, human serum albumin (HSA) as the major transporter of fatty acids was modified with glyoxal under physiological conditions. Reversibly albumin-bound glyoxal was removed, and advanced glycation end products were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total modification of protein-bound lysine and arginine residues reached up to 4.2 and 9.6%, respectively. The impact of these modifications on the transport capacity of long-chain fatty acids was characterized by spin-labeled fatty acid probes via electron paramagnetic resonance spectroscopy. With increasing degree of glycation, the equivalence of the seven binding sites of native HSA with a dissociation constant of 0.74 ± 0.09 μM was set off with only the three high-affinity sites 2, 4, and 5 remaining (0.46 ± 0.07 μM). The other four sites were shifted to low affinities with significantly higher dissociation constants (1.32 ± 0.35 μM). Tryptic peptide mapping enabled us to relate these findings to molecular changes at specific binding sites. Modification hotspots identified were lysine 351, 286, 159 and arginine 144, 485, 117. Further investigation of plasma protein samples of uremic patients vs healthy controls gave first insights into the in vivo situation.

Single-Cell Profiling of Fatty Acid Uptake Using Surface-Immobilized Dendrimers.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Abstract 1266: Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy

Abstract Dysregulated fatty acid metabolism is thought to be a hallmark of cancer, wherein fatty acids function both as an energy source and as signals for enzymatic and transcriptional networks contributing to malignancy. Fatty acid-binding protein 5 (FABP5) is an intracellular protein that facilitates transport of fatty acids and plays a role in regulating the expression of genes associated with cancer progression such as cell growth, survival, and metastasis. Overexpression of FABP5 has been reported to contribute to an aggressive phenotype and a poor survival correlation in several cancers. Therefore, inhibition of FABP5 is considered as a therapeutic approach for cancers. Phenotypic screening of a library of covalent compounds for selective sensitivity of cancer cells followed by medicinal chemistry optimization resulted in the identification of AUR104 with desirable properties. Chemoproteomic-based target deconvolution revealed FABP5 as the cellular target of AUR104. Covalent adduct formation with Cys43 of FABP5 by AUR104 was confirmed by mass spectrometry. Target occupancy studies using a biotin-tagged AUR104 demonstrated potent covalent binding to FABP5 in both cell-free and cellular conditions. Ligand displacement assay with a fluorescent fatty acid probe confirmed the competitive binding mode of AUR104 with fatty acids. Binding at the fatty acid site and covalent bond formation with Cys43 were also demonstrated by crystallography. Furthermore, AUR104 showed a high degree of selectivity against a broad safety pharmacology panel of enzymes and receptors. AUR104 exhibited potent anti-proliferative activity in a large panel of cell lines derived from both hematological and solid cancers with a high degree of selectivity over normal cells. Anti-proliferative activity in lymphoma cell lines correlated with inhibition of MALT1 pathway activity, cleavage of RelB/Bcl10 and secretion of cytokines, IL-10 and IL-6. AUR104 displayed desirable drug-like properties and dose-dependent oral exposure in pharmacokinetic studies. Oral dosing with AUR104 resulted in dose-dependent anti-tumor activity in DLBCL (OCI-LY10) and NSCLC (NCI-H1975) xenograft models. In a repeated dose MTD studies in rodents and non-rodents, AUR104 showed good tolerability with an exposure multiple of >500 over cellular EC50 for up to 8 hours. In summary, we have identified a novel covalent FABP5 inhibitor with optimized properties that showed anti-tumor activity in in vitro and in vivo models with acceptable safety profile. The data presented here strongly support clinical development of AUR104. Citation Format: Dinesh Chikkanna, Leena Khare Satyam, Sunil Kumar Pnaigrahi, Vinayak Khairnar, Manoj Pothuganti, Lakshmi Narayan Kaza, Narasimha Raju Kalidindi, Vijaya Shankar Nataraj, Aditya Kiran Gatta, Narasimha Rao Krishnamurthy, Sandeep Patil, DS Samiulla, Kiran Aithal, Vijay Kamal Ahuja, Nirbhay Kumar Tiwari, KB Charamannna, Pravin Pise, Thomas Anthony, Kavitha Nellore, Sanjeev Giri, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1266.

A Distal Loop Controls Product Release and Chemo- and Regioselectivity in Cytochrome P450 Decarboxylases.

Cytochrome P450 OleT utilizes hydrogen peroxide (H2O2) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron-oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F-G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F-G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F-G loop is essential for regulating regiospecific C-H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid.

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C-C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C-C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Visualizing Wnt Palmitoylation in Single Cells.

Wnt palmitoylation regulates its secretion and signaling activity in cells. Methods to monitor cellular Wnt palmitoylation are instrumental in investigating Wnt activity, secretion, and its interaction with cellular membrane compartments. This protocol describes a method we have recently developed to detect cellular Wnt palmitoylation. The method, combining click chemistry, bio-orthogonal fatty acid probes, and proximity ligation assay (PLA), provides high sensitivity and subcellular resolution for detection of Wnt palmitoylation. It is also compatible with multiple imaging platforms, and is applicable to detecting palmitoylated forms of other fatty acylated proteins.

Unbiased Clustering of Molecular Dynamics for Spatially Resolved Analysis of Chemically Heterogeneous Surfaces.

A technique is described for resolving and interpreting molecular interactions with a chemically heterogeneous surface. Using total internal reflection fluorescence microscopy, dynamic single molecule trajectories were accumulated simultaneously for fluorescently labeled fatty acid (interacting primarily via hydrophobic interactions) and dextran (interacting via hydrogen-bonding interactions) probe molecules at the interface between an aqueous solvent and a photopatterned solid surface with distinct regions of amine-terminated and poly(ethylene glycol) self-assembled monolayers. Using dynamic properties of the probe molecules (adsorption rate, surface diffusion coefficient, residence time), an unsupervised Gaussian mixture model algorithm was used to identify areas of the surface that were chemically related to each other, and the dynamic behaviors of the probe molecules were studied statistically on these distinct regions. The dynamic data were compared to data from homogeneous surfaces of known chemistry to provide a chemical identification of each location on the surface. Spatial maps were also constructed, allowing for spatial visualization of surface chemistry on a hydrophilic substrate. This work enables the direct study of interactions between single-molecule probes and distinct surface chemistries, even in the presence of spatial heterogeneity, without human bias, assumptions about surface structure, or model-dependent analysis.

Specific Ion (Hofmeister) Effects on Adsorption, Desorption, and Diffusion at the Solid–Aqueous Interface

Certain salts in aqueous solution are known to significantly increase the affinity of molecules with hydrophobic moieties for a hydrophobic interface, but the mechanism of this effect is poorly understood. We used single-molecule total internal reflection fluorescence microscopy to directly observe the underlying dynamic interfacial phenomena for a fluorescent fatty acid probe molecule at a self-assembled monolayer surface. Both NaF and NaSCN increased the surface affinity of the probe from that observed in pure water, and consistent with expectations, the surface affinity was greater in the presence of NaF than that with NaSCN. Notably, the primary cause of the increase in surface affinity was specifically due to an increase in the absolute rate of adsorption from solution. In contrast, the surface residence time and the surface diffusion coefficient of the probe molecules did not depend significantly on the solution conditions.

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis.

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function.

Fluorescent probes as a tool for labelling and tracking the amphibian chytrid fungus Batrachochytrium dendrobatidis

The dissemination of the virulent pathogen Batrachochytrium dendrobatidis (Bd) has contributed to the decline and extinction of many amphibian species worldwide. Several different strains have been identified, some of which are sympatric. Interactions between co-infecting strains of a pathogen can have significant influences on disease epidemiology and evolution; therefore the dynamics of multi-strain infections is an important area of research. We stained Bd cells with 2 fluorescent BODIPY fatty acid probes to determine whether these can potentially be used to distinguish and track Bd cell lines in multi-strain experiments. Bd cells in broth culture were stained with 5 concentrations of green-fluorescent BODIPY FL and red-fluorescent BODIPY 558/568 and visualised under an epifluorescent microscope for up to 16 d post-dye. Dyed strains were also assessed for growth inhibition. The most effective concentration for both dyes was 10 pM. This concentration of dye produced strong fluorescence for 12 to 16 d in Bd cultures held at 23 degrees C (3 to 4 generations), and did not inhibit Bd growth. Cells dyed with BODIPY FL and BODIPY 558/568 can be distinguished from each other on the basis of their fluorescence characteristics. Therefore, it is likely that this technique will be useful for research into multi-strain dynamics of Bd infections.

Soil methane oxidation and methanotroph responses to afforestation of pastures with Pinus radiata stands

Afforestation of pastures in New Zealand reduces methane (CH 4) production from soil, while also stimulating oxidation of atmospheric CH 4 by soil methanotrophs. However, neither the mechanisms by which soil CH 4 oxidation is enhanced by afforestation, nor how long after forest planting tree-dependent responses in CH 4 oxidation become detectable are fully known. Here, we investigated the effects of different-aged stands (5–20 y) of the exotic pine ( Pinus radiata (D. Don)) on CH 4 oxidation and methanotrophic community structure in soils, compared with adjacent, long-established pastures. Two of the pastures were on volcanic soils and two were on non-volcanic soils. Although the CH 4 fluxes in soils from these young stands were not significantly different from those in the associated pastures, the rate of oxidation of added 13CH 4 was higher in the pine soils. Both fluxes and 13CH 4 oxidation rates were higher in the volcanic than the non-volcanic soils. Combined phospholipid fatty acid (PLFA) and stable isotope probe (SIP) analyses suggested that type II methanotrophs (PLFA C18:1ω7) were most active in all soils followed by uncultivable bacteria (C17:0ai). Molecular analysis of the methanotrophic community structure using pmoA (particulate methane monooxygenase) genes suggested that a particular type II methanotroph (TRF 35) was dominant in all soils, but more so in the pine than in pasture soils. A type I methanotroph (TRF 245) was more prevalent in the pasture than in associated pine soils, whereas TRF 128 (a type II methanotroph) was slightly more dominant in soils under pine. Cloning and sequencing data suggest TRFs 35 and 128, which differ from one another, belong to distant relatives of Methylocapsa sp; TRF 245 is related to Methylococcus capsulatus. Land-use change resulted in changes in soil bulk density, porosity, moisture contents and in methanotrophic community structure. Methane oxidation rates were most closely related to soil moisture, as well as to the methanotrophic community structure, and nitrate-N, extractable C and total C concentrations. Stepwise multiple regression also suggested a weak effect ( P = 0.06) of stand age on CH 4 oxidation rate. By contrast, the responses of the methanotrophic community structure to this land-use change were more readily detected by the specific molecular analyses, and indicated a predominance of type II methanotrophs in pine soils.

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl- l-α phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.

Investigation of SDS, DTAB and CTAB micelle microviscosities by electron spin resonance

Electron spin resonance spectroscopy (ESR) of the nitroxide labelled fatty acid probes (5-, 16-doxyl stearic acid) was used to monitor the micelle microviscosity of three surfactants at various concentrations in aqueous solution: sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). At low surfactant concentration, there is no micelle, the ESR probe is dissolved in water/surfactant homogeneous phase and gives his microviscosity. At higher surfactant concentration, an abrupt increase in microviscosity indicates the apparition of micelles and, the solubilization of the probes in micelles. The microviscosity of the three surfactants, in a large surfactant range, was obtained as well as the critical micelle concentration (CMC). The microviscosity increased slightly with the increase in surfactant concentration. Phosphate buffer lowered the CMC value and generally increased the microviscosity.

Titration of Fatty Acids Solubilized in Cationic and Anionic Micelles. Calorimetry and Thermodynamic Modeling

The electrostatic properties of charged surfactant micelles are investigated through titrations of fatty acid probes solubilized in the micelles. The titration process is followed by means of calorimetric measurements and by determining the pH values as a function of added base. This approach yields a complete thermodynamic description of the titration process. In particular, we find that the process is endothermic at 298 K. This is contrary to the titration of carboxylic acids in water, where DeltaH is approximately 0. To identify the main effect underlying the difference in DeltaH between titration in a micelle and water, a thermodynamic model has been developed which focuses on the transfer properties of charged and uncharged species from bulk water to the surface of a micelle and which incorporates a dielectric discontinuity at the micellar surface. The model relies on the use of the Poisson-Boltzmann equation which is solved using a finite element method. Experimental results and the model calculations imply that the dielectric discontinuity at (or near) the micellar surface plays a major role and hence must be included when analyzing the titration behavior of an acid functionality at the surface of a charged micelle.