Interaction of fluorinated ether anesthetics with artificial membranes

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T₁) measurements are consistent with substantial immobilization of the lipid-bound anesthetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The ¹⁹F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the ¹⁹F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, ¹⁹F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo ¹⁹F-NMR surface coil and imaging studies.     

Measurement of transmembrane pH gradients in human erythrocytes using 19F NMR

A new high-sensitivity method has been described for measuring transmembrane pH gradients in vesicular systems using ¹⁹F NMR. The ¹⁹F resonance of trifluoroethylamine has been shown to have a large pH-dependent chemical shift and the position of the resonance was measured with high precision and sensitivity. In suspensions of human erythrocytes, trifluoroethylamine distributed itself across the membrane and separate ¹⁹F resonances were obtained from the trifluoroethylamine inside and outside of the cells. The pH in each compartment was calculated from the resonance positions.     

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by ¹H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from ¹H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the ¹H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-of-saturation by cross-relaxation method.     

Interaction of fluorinated ether anesthetics with artificial membranes.

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

113Cd nmr study of the metal cluster structure of human liver metallothionein

Cadmium-113 nuclear magnetic resonance (¹¹³Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled ¹¹³Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for ¹¹³CdCl₂ during isolation. The two isoproteins, MT-1 and MT-2, showed ¹¹³Cd nmr resonances in the chemical shift range 610–670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent ¹¹³Cd ions linked by cysteine thiolate ligands. Homonuclear ¹¹³Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the ¹¹³Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.     

Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and ¹⁹F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The ¹⁹F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

In vivo 19F-NMR study of isoflurane elimination from brain

The time course of isoflurane elimination from rabbit brain was studied in vivo with 19F-NMR spectroscopy. Two exponential decay functions with different time constants were observed and assigned to two distinct brain compartments. Isoflurane has a 26 min time constant for one compartment (similar to a value of 25 min with halothane) but 174 min in the second one, compared with 320 min for halothane. The shorter half-life for isoflurane may be due to lower solubility of this agent in brain tissue. Comparison of isoflurane 19F chemical shifts in solvents in isolated brain lipids and in whole brain tissue indicates that the anesthetic present in the brain exists in a single environment (on the NMR time scale), which is a weighted average of both hydrophilic and hydrophobic environments.     

Interfacial adsorption of an inhalation anesthetic onto ionic surfactant micelles and its desorption by high pressure

The effects of pressure and temperature on the critical micelle concentration (CMC) of sodium dodecylsulfate (SDS) were measured in the presence of various concentrations of an inhalation anesthetic, methoxyflurane. The change in the partial molal volume of SDS on micellization, $\text{Δ}\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, increased with the increase in the concentration of methoxyflurane. The CMC-decreasing power, which is defined as the slope of the linear plot between ln(CMC) vs. mole fraction of anesthetic, was determined as a function of pressure and temperature. Since the CMC-decreasing power is correlated to the micelle/water partition coefficient of anesthetic, the volume change of the transfer (ΔV_p^o) of methoxyflurane from water to the micelle can be determined from the pressure dependence of the CMC-decreasing power. The value of ΔV_p^o amounts 6.5±1.8 cm³·mol^?1, which is in reasonable agreement with the volume change determined directly from the density data, 5.5±0.6 cm³ · mol^?1. Under the convention of thermodynamics, this indicates that the application of pressure squeezes out anesthetic molecules from the micelle. The transfer enthalpy of anesthetic from water to the micelle is slightly endothermic. The partial molal volume of methoxyflurane in the micelle (112.0 cm³·mol^?1) is smaller than that in decane (120.5 cm³·mol^?1) and is larger than that in water (108.0 cm³·mol^?1). This indicates that the anesthetic molecules are incorporated into the micellar surface region, i.e., the palisade layer of the micelle in contact with water molecules, rather than into the micelle core.     

Approaches for the measurement of solvent exposure in proteins by <Superscript>19</Superscript>F NMR

Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of ¹⁹F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, ¹⁹F spin–lattice relaxation rates, ¹H–¹⁹F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen. Both the solvent isotope shifts and paramagnetic shifts from dissolved oxygen sensitively reflect solvent exposed surface areas.     

Quantum mechanical calculations and experimental measurement of N-terminal charge effects on 1HN and 1HCα chemical shifts in peptides

Nuclear magnetic resonance spectroscopists are increasingly utilizing chemical shifts to characterize the secondary structure of proteins. The present study addresses the effects that the positively charged amino group at the N-terminus of a peptide has on ¹HN and ¹HCα chemical shifts along the chain. This information is necessary for interpreting chemical shift data for proteins and/or for peptides that are used as models for protein structure. The chemical shifts for the ¹H resonances of four peptides that differ only in the location of their N-terminii are assigned using two-dimensional nmr spectroscopy. The peptides have sequences derived from the β subunit of the glycoprotein hormone human chorionic gonadotropin (hCG-β). Comparison of the ¹HN and the ¹HCα chemical shifts for residues common to all four peptides reveals downfield shifts for ¹HN and the ¹HCα resonances within three residues of the N-terminus compared with chemical shifts in the interior of the peptide. The magnitude of the downfield shift is larger for resonances nearer the N-terminus. Quantum mechanical calculations of the ¹HN and ¹HCα chemical shifts in peptides constructed with six alanine units also predict a significant terminus effect. The calculations agree both qualitatively and quantitatively with the experimental data. The inductive nature of the end effect is confirmed in the calculations by Mulliken population analysis. End effects should be taken into account in determining protein secondary structures from chemical shifts. © 1996 John Wiley & Sons, Inc.     

A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes

The binding of methylmercury, CH₃Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. To suppress or completely eliminate interfering resonances from the much more abundant hemoglobin protons, spectra were measured by a technique based on the transfer of saturation throughout the envelope of hemoglobin resonances following a selective presaturation pulse or by the spin-echo Fourier transform method. With these techniques, ¹H-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, in both intact and hemolyzed erythrocytes to which CH₃Hg(II) had been added. The results for intact erythrocytes indicate that part of the CH₃Hg(II) is complexed by intracellular glutathione. These results also indicate that exchange of CH₃Hg(II) among glutathione molecules is fast, with the average lifetime of a CH₃Hg(II)-glutathione complex estimated to be less than 0.01 s. From exchange-averaged chemical shifts of the resonance for the proton on the α-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the sulfhydryl group of glutathione binds CH₃Hg(II) more strongly than the sulfhydryl groups of hemoglobin.     

F NMR studies of solvent exposure and peptide binding to an SH3 domain

¹⁹F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct ¹⁹F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. ¹⁹F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. ¹⁹F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH₂, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the ¹⁹F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, k_ex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s^− 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of ¹⁹F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents.     

In vivo 19F-NMR study of halothane distribution in brain

Halothane distribution and elimination from rabbit brain was studied in vivo using 19F-NMR spectroscopy. Two exponential decay functions for the anesthetic were observed in the clearance curve. They are assigned to halothane in brain held in two distinct chemical environments characterized by different chemical shifts, and half-lives (25 and 320 min). A nonvolatile halothane metabolite with a half-life of several days was found to be present in rabbit brains. The in vivo results were corroborated by ex vivo experiments on excised brain tissue. Halothane was distributed in all of the major cell subfractions, whereas the metabolite was present predominantly in the cytoplasm.     

Noninvasive In Vivo Demonstration of 2-Fluoro-2-Deoxy-d-Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.     

19F-NMR of fluorine labeled acyl chains in phospholipid bilayer vesicles

¹⁹F-labeled phospholipids, L-α-bis-(ω-fluoro palmitoyl)phosphatidylcholine, L-α-bis-(12,12-difluoro stearoyl)phosphatidylcholine and L-α-bis-(6,6-difluoro palmitoyl)phosphatidylcholine, were incorporated in phospholipid vesicles by sonication of aqueous lipid emulsions. Vesicles were prepared both from the pure fluorine substituted phospholipids as well as from lipid mixtures obtained by combining the fluorine substituted lipids with the synthetic phospholipids, L-α-dilauroyl-, L-α-dimyristoyl-, L-α-dipalmitoyl- and L-α-distearoyl-phosphatidylcholine. Characterization by gel permeation chromatography showed that stable unilamellar vesicles with diameters of ≌200 Å could be obtained with a minimum or absence of multilamellar material. The vesicles give rise to two ¹⁹F resonances in most cases as observed previously [K.J. Longmuir and F.W. Dahlquist, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2716]. The chemical shift differences undergo systematic changes that confirm the interpretations that the dual ¹⁹F resonances arise from the inner and outer halves of the vesicle bilayer. The shift separation increases systematically as the fluorine label is positioned closer to the phospholipid headgroup and decreases systematically with increasing temperature. Both observations agree with what is currently known about phospholipid vesicle structure. Anomalous results are obtained with DSPC as host vesicle since only a single resonance of inbedded fluorinated phospholipids is found.     

NMR study of general anesthetic interaction with nAChR beta2 subunit

The molecular basis of anesthetic interaction with membrane proteins has been explored via determination of anesthetic effects on the structure and dynamics of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) β₂ subunit in dodecylphosphocholine (DPC) micelles by ¹H and ¹⁵N solution-state NMR. Both 1-chloro-1,2,2-trifluorocyclobutane (F3) and isoflurane, two volatile general anesthetics, induced nonuniform changes in chemical shifts among residues in TM2e. Saturation transfer difference NMR experiments further confirmed the direct anesthetic interaction with TM2e. A significant and more specific anesthetic interaction was observed on three leucine residues at the helix C-terminus. Although the TM2e helical structure remained after addition of anesthetics, plausible shortening and lengthening of helix hydrogen bonds were evidenced by periodic changes in backbone amide chemical shifts. The TM2e backbone dynamics were determined on the basis of the ¹⁵N relaxation rate constants, R₁ and R₂, and the ¹⁵N-[¹H] NOE using the model-free approach. The global tumbling time (11.7 ns) of TM2e in micelles slightly increased (∼12.3-12.5 ns) in the presence of anesthetics. The order parameter, S², exceeded 0.9 for all ¹⁵N-labeled residues, showing a restricted internal motion. Anesthetics appear to have minor effect on the TM2e's internal motion. This study provided the basis for subsequent more comprehensive studies of anesthetic effects on the transmembrane domain complex of neuronal nAChR.     

Tautomeric composition of D-fructose phosphates in solution by fourier transform carbon-13 nuclear magnetic resonance

The Fourier transform ¹³C magnetic resonance spectra of D-fructose 6-phosphate (F6P) and D-fructose 1,6-diphosphate (FDP) were obtained. The signal assignments made on the basis of ¹³C chemical shifts and ¹³C-³¹P spin-spin couplings indicate that the earlier assignments of the C-4 and C-5 resonances of α- and β-fructofuranose in oligosaccharides and D-fructose [Allerhand, A. and Doddrell, D., J. Amer. Chem. Soc., $\text{93}$, 2777, 2779 (1971)] should be reversed. Integration of signal intensities yields the following equilibrium composition at 35°C: F6P, α-anomer 19±2% and β-anomer 81±2%, FDP, α-anomer 23±4% and β-anomer 77±4%. Less than 1.5% keto or hydrated keto form is present in solutions of either fructose phosphate. The bearing of these findings on the tautomeric specificity of phosphofructokinase is discussed.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR

Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.