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At least 19 recordsLinked to original sources
PubMed1998

Inhibition of excitatory amino acid-activated currents by trichloroethanol and trifluoroethanol in mouse hippocampal neurones.

1. The effects of the active metabolite of chloral derivative sedative-hypnotic agents, 2,2,2-trichloroethanol (trichloroethanol), and its analog 2,2,2-trifluoroethanol (trifluoroethanol), were studied on ion current activated by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents in a concentration-dependent manner. Trichloroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 6.4 and 12 mM, respectively, while trifluoroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 28 and 35 mM, respectively. 3. Both trichloroethanol and trifluoroethanol appeared to be able to inhibit excitatory amino acid-activated currents by 100 per cent. 4.Concentration-response analysis of NMDA- and kainate-activated current revealed that trichloroethanol decreased the maximal response to both agonists without significantly affecting their EC50 values. 5. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents more potently than did ethanol. The inhibitory potency of trichloroethanol and trifluoroethanol appears to be associated with their increased hydrophobicity. 6. The observation that trichloroethanol inhibits excitatory amino acid-activated currents at anaesthetic concentrations suggests that inhibition of excitatory amino acid receptors may contribute to the CNS depressant effects of chloral derivative sedative-hypnotic agents.

Animals↗
PubMed2002

Dissecting the effect of trifluoroethanol on ribonuclease A. Subtle structural changes detected by nonspecific proteases.

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 degrees C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.

Circular Dichroism↗
PubMed1994

Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.

The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.

Amino Acid Sequence↗
PubMed1998

Inactivation and conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions.

Conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions have been followed by fluorescence emission and circular dichroism spectroscopy. At low concentration (less than 5%), trifluoroethanol shows a reversible inhibition competitive to ethanol and noncompetitive to NAD+. The inhibition constants for native and structural-zinc-removed yeast alcohol dehydrogenase were 5.8 and 1.1 mM, respectively, suggesting that the active site becomes more flexible after the structural zinc is removed. At higher trifluoroethanol concentrations the enzyme was irreversibly inactivated. Comparison of inactivation and conformational changes of yeast alcohol dehydrogenase denatured in trifluoroethanol solutions shows that the extent of inactivation is larger than the extent of conformational changes at the same trifluoroethanol concentration. The results obtained from circular dichroism spectra show that the presence of trifluoroethanol can induce the formation of secondary structure of the enzyme.

Alcohol Dehydrogenase↗
PubMed1985

Early stages in the trifluoroethanol-induced unfolding of hen egg-white lysozyme and its complex with (GlcNAc)3.

The trifluoroethanol-induced unfolding of hen egg-white lysozyme was studied by circular dichroism. It was shown that if the H2O/trifluoroethanol ratio is above 10:1 (v/v), the unique three-dimensional structure of the protein is not affected, whereas within the ration 10:1-2.8:1 (v/v), this structure is partially unfolded. At the ratio 2.4:1 (v/v), the native conformation of lysozyme is completely disrupted and the conformational transition fits a two-state model. A similar effect was observed for the trifluoroethanol-induced unfolding of the lysozyme-(GlcNAc)3 complex. Within the H2O2 trifluoroethanol ratio 15:1-5.5:1 (v/v), the characteristic intensities of the Cotton effects which arise from the association of (GlcNAc)3 with the active site of lysozyme, diminished and approached those exhibited by lysozyme itself at the same H2O trifluoroethanol ratios. This shows that (GlcNAc)3 is released from the protein surface in early stages of the unfolding process. At the ratio 2.4:1 (v/v), the lysozyme-(GlcNAc)3 complex was completely disrupted and the protein unfolded. It is suggested that a considerable alteration in hydration of the lysozyme molecule caused by trifluoroethanol increases protein surface fluctuations, causing the release of (GlcNAc)3 from the active site of lysozyme.

Acetylglucosamine↗
PubMed2002

Change of mobile phase pH during gradient reversed-phase chromatography with 2,2,2-trifluoroethanol-water as mobile phase and its effect on the chromatographic hydrophobicity index determination.

We have shown previously that using a trifluoroethanol containing mobile phase provides a unique chromatographic selectivity. This is essential to derive molecular descriptors by HPLC which requires retention data from several systems. It also requires that the ionisation is suppressed so that retention times reflect the properties of the neutral molecules. Therefore the pH change of the mobile phase during gradient elution and its effect on the solute ionisation have been studied. During gradient elution of mixtures of ammonium acetate and butylammonium formate with trifluoroethanol as an organic modifier it was found that the pH was almost constant when the gradient started with a low pH. However, when the starting mobile phase pH was above 8 the pH dropped very quickly as the trifluoroethanol concentration increased in the mobile phase. The CHI descriptor (a retention index derived directly from gradient retention times) of several basic compounds as a function of starting mobile phase pH has been measured using trifluoroethanol gradient. The effect of the trifluoroethanol on the pKa change of the compounds has been investigated. The experimental data fit closely to a previously derived equation that describes gradient retention times as a function of mobile phase pH and analyte ionisation constant (pKa). This equation makes it possible to predict the CHI descriptor for ionisable compounds at various pH values. We have used butylamine for high pH mobile phase preparation as is more basic than ammonia and for many basic drugs the retention of the neutral form could be obtained directly (without extrapolation).

Buffers↗
PubMed1997

Conformational changes of creatine kinase in trifluoroethanol solutions.

The conformational changes of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) in trifluoroethanol solutions have been followed by fluorescence emission and circular dichroism spectra. At low trifluoroethanol concentrations, less than 15%, the enzyme was completely inactivated with no observed marked conformational changes. The fluorescence emission maximum of the native enzyme was at 337 nm. With increasing trifluoroethanol concentration, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 40% trifluoroethanol, indicating that the tryptophan residues were completely exposed. The results obtained from CD spectra show that the presence of trifluoroethanol can induce the formation of secondary structure in the native enzyme and in urea-denatured enzyme.

Binding Sites↗
PubMed1996

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.

Adrenocorticotropic Hormone↗
PubMed2003

The solution structures and activity of caerin 1.1 and caerin 1.4 in aqueous trifluoroethanol and dodecylphosphocholine micelles.

The caerin 1 peptides are among the most powerful of the broad-spectrum antibiotic amphibian peptides. Caerin 1.1 has previously been shown to form an amphipathic helix-bend-helix structure in aqueous trifluoroethanol (H. Wong, J. H. Bowie, and J. A. Carver European Journal Biochemistry, 1997, Vol. 247, pp. 545-557) and structure-activity relationship studies indicate that both helices are required for activity, as well as flexibility in the bend region connecting the two. The structure of caerin 1.1 in dodecylphosphocholine micelles was investigated and shown to be very similar to that determined in aqueous trifluoroethanol. Caerin 1.4, which is identical to caerin 1.1, but with serine residues replacing Val5 and Gly7, is less active than caerin 1.1 against most bacterial species but has improved activity against Escherichia coli and Micrococcus luteus. The solution NMR structure of caerin 1.4 was determined in both aqueous trifluoroethanol and dodecylphosphocholine micelles, and was shown to be similar to caerin 1.1. It was concluded that differences in the hydrophobicity and hydrophilic angle of the first helix are probably responsible for the different spectra of antibacterial activity. The similarity of the structures calculated in aqueous trifluoroethanol and dodecylphosphocholine micelles suggests that, for caerin 1.1 and 1.4, these solvent systems are equally as good at representing a membrane environment.

Amino Acid Sequence↗
PubMed1996

Sensory irritation mechanisms investigated from model compounds: trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether.

Quantitative structure-activity relationships (QSAR) have suggested the importance of hydrogen bonding in relation to activation of the sensory irritant receptor by nonreactive volatile organic chemicals. To investigate this possibility further, three model compounds with different hydrogen bond acidity, trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether, were selected for study. The potency of each chemical is obtained from the concentration necessary to reduce respiratory rate in mice by 50% (RD50). The RD50 values obtained were: methyl hexafluoroisopropyl ether (> or = 160,000 ppm), trifluoroethanol (11,400-23,300 ppm), and hexafluoroisopropanol (165 ppm). QSAR showed that trifluoroethanol and methyl hexafluoroisopropyl ether behaved as predicted as nonreactive sensory irritants, whereas hexafluoroisopropanol was much more potent than predicted. The higher than predicted potency of hexafluoroisopropanol could be due to a coupled reaction, involving both strong hydrogen bonding and weak Brönsted acidity. A concerted reaction could thus be more efficient in activation of the receptor. Hydrogen bonding properties and concerted reactions may be important in the activation of the sensory irritant receptor by nonreactive volatile organic chemicals.

1-Propanol↗
PubMed2001

Unique selectivity of perfluorinated stationary phases with 2,2,2-trifluoroethanol as organic mobile phase modifier.

The selectivity of Luna C18 Xterra C18 and Fluophase (perfluorinated C6) stationary phases has been investigated with aqueous acetonitrile, methanol and 2,2,2-trifluoroethanol mobile phases using linear solvation equations. The gradient retention times of a set of 60 compounds with known molecular descriptors have been determined. Linear solvation equations have been set up to describe the relationship between the gradient retention times and the molecular properties. The selectivity of the stationary phase/mobile phase systems was characterised by the regression coefficients of the molecular descriptors. The perfluorinated stationary phase showed very different selectivity using 2,2,2-trifluoroethanol (TFE) as co-solvent. Compounds with H-bond donor functionality were retained much less than in the other investigated high-performance liquid chromatography (HPLC) systems. This unique selectivity can be explained by the stronger adsorption of trifluoroethanol on the perfluorinated stationary phase surface, than on the hydrocarbon surface. It suggests the importance of the adsorbed organic modifiers in the separation mechanism during reversed-phase HPLC.

Chromatography, High Pressure Liquid↗
PubMed2001

Molecular dynamics simulation of the antimicrobial salivary peptide histatin-5 in water and in trifluoroethanol: a microscopic description of the water destructuring effect.

The results of 520 ps molecular dynamics simulation of histatin-5, a small peptide present in human saliva and possessing antimicrobial activity, dissolved in water and in 2,2,2-trifluoroethanol, are reported. The simulations indicate that histatin-5 is destabilized in water and begins to unfold after 250 ps, while in organic solvent it maintains a regular secondary structure throughout the trajectory. Analysis of the peptide-solvent hydrogen bonds indicates that 2,2,2-trifluoroethanol is a poorer proton acceptor than water. The fluorine atom of the alcohol is almost never engaged in a hydrogen bond and the organic solvent interacts mainly with the peptide through its hydroxyl group. For some residues analysis of the solvent residence time indicated longer values for 2,2,2-trifluoroethanol than for water. The most striking difference is related to the number of times the solvent enters and leaves the first coordination shell of the peptide. This value was more than one order of magnitude higher for water than for the alcohol, suggesting that this may be the main cause of alpha-helix destabilization perpetrated by water.

Amino Acid Sequence↗
PubMed1978

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.

Acetaldehyde↗
PubMed1995

Effect of pH on the conformation and backbone dynamics of a 27-residue peptide in trifluoroethanol. An NMR and CD Study.

The C-terminal fragment, residues 385-411, from human fibrinogen gamma-chain, i.e. KIIPFNRLTIGEGQQHHLG-GAKQAGDV, shows multiple turn conformations in aqueous solution (Mayo, K. H., Burke, C., Lindon, J. N., and Kloczewiak, M. (1990) Biochemistry 29, 3277-3286). The present study investigates the effect of pH and trifluoroethanol on the conformation and backbone dynamics of this 27-residue peptide. Both circular dichroism (CD) and 1H-NMR data indicate the normally observed increased helical conformations as a function of increasing trifluoroethanol. 1H-NMR structural studies done in the presence of 40% trifluoroethanol, pH 5.3, yield a network of nuclear Overhauser effects consistent with significant populations of helix-like conformation. Distance geometry calculations based on nuclear Overhauser effect-derived distance constraints yield a family of structures with relatively well defined N- and C-terminal conformations and an ill defined mid-peptide region from Gly397 to Gly403. Similar conformational populations are observed at pH 2.5. CD studies, however, indicate an increase in average alpha-helix content on decreasing the pH from 6 to 2. This apparent conflict between CD and NMR results may be explained by a transition from multiple beta-turn character at pH 5.3 to increased alpha-helix structure at pH 2.5. 13C alpha NMR relaxation data analyzed with the Lipari-Szabo model-free approach provide order parameters that demonstrate little if any influence of pH on backbone motional restrictions within the more flexible mid-peptide domain. At low pH, however, motions become less restricted within N-terminal residues Lys385-Phe389 and more restricted within C-terminal residues Ala405-Val411.

Amino Acid Sequence↗
PubMed1995

Limited proteolysis of lysozyme in trifluoroethanol. Isolation and characterization of a partially active enzyme derivative.

Proteolysis of hen egg-white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6-24 h at 40-52 degrees C produces a 'nicked' protein species which was purified to homogeneity by reverse-phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129-residue chain of lysozyme at peptide bond Lys97-Ile98. Nicked lysozyme, which is therefore constituted by fragments 1-97 and 98-129 cross-linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25 degrees C or 5 degrees C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28 degrees C in comparison with 73 degrees C for the unmodified enzyme, both at pH 7.0. Inspection of the X-ray structure of hen lysozyme reveals that thermolysin cleaves at the C-terminus of alpha-helix C (residues 88-98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50% aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native-like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669-678]. Thus, whereas native lysozyme in its well-packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all-or-none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.

Amino Acid Sequence↗
PubMed1977

Circular dichroism of insulin A chain in water: trifluoroethanol mixtures. Use of linear and nonlinear least squares analysis.

Circular dichroic spectra obtained for S-carboxymethylated insulin A chain in water: trifluoroethanol mixtures show that a marked conformational transition occurs as the concentration of trifluoroethanol is increased to (v/v) 83% to give a polypeptide containing about 43% of the residues in the alph-helical conformation. Several proposed methods of analysis, including both linear and two nonlinear lest squares methods, were unable to quantitate the amount of beta-structure present in the polypeptide in 83% trifluoroethanol. Examination of the methods of analysis lead to the conclusion that the current models for far UV CD analysis are not adequare for the data obtained in this study and shows that nonlinear least squares procedures may lead to erroneous conclusions.

Circular Dichroism↗
PubMed2003

Capillary electrophoretic study of the binding of zinc(II) ion to bacitracin A1 in water-2,2,2-trifluoroethanol.

Binding of Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis in water/2,2,2-trifluoroethanol (70/30 v/v) at different apparent pH values in order to estimate the association constant of metal, the acidic dissociation constants and the Stokes radii of both free and bounded peptide in apolar environment. The Stokes radii of the free peptide species were compared with those in aqueous solution, as obtained in a recent study performed by our group, indicating that apolar environment stabilizes bacitracin A(1) in a conformational structure with the lateral chain of apolar amino acids exposed on the external surface. This conformation of the macrocyclic dodecapeptide is ready to interact with Zn(2+) ion, as pointed out by the strong increase of the association constant measured in water/2,2,2-trifluoroethanol with respect to the value obtained in aqueous solution. In addition, whereas Zn(2+) ion binding in aqueous solution provides a sensible reduction of peptide Stokes radius, no sensible variations following to ion binding were observed in hydro-organic solution. The present results suggest that the apolar environment, rather than the metal ion binding, could be responsible for the conformational transition that brings bacitracin A(1) towards its biologically active structure.*

Bacitracin↗
PubMed1998

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1.

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.

Amino Acid Sequence↗