Nucleotide sequence of nuclear 5.7S RNA of mouse cells

Viable tumor cells were examined by ¹⁹F-NMR spectroscopy after treatment with 4-trifluoromethyl 2,6-dinitrophenyl sulphonate (CF₃-DNBS), which is an analog of 2,4,6-trinitrophenyl sulphonate (TNBS). The presence of a strong ¹⁹F-NMR signal from treated cells suggested the binding of the “probe”. Treatment of labelled cells with proteolytic enzymes significantly decreased the signals, suggesting that the label was essentially bound to the cell surface macromolecules. A proportion of the material bound to the cell was removable by dialysis of cell extracts against a structural analog, suggesting that some CF₃-DNBS-cell membrane bonds were not covalent. The existence of such non-covalent bonds has also been confirmed with soluble proteins. By the same approach, it was also found that ¹⁴C-TNBS formed covalent and non-covalent bonds with tumor cells.     

Evaluating the amino acid CF3-bicyclopentylglycine as a new label for solid-state 19 F-NMR structure analysis of membrane-bound peptides.

The conformation, alignment and dynamic behavior of membrane-bound peptides is readily accessible by solid-state (19)F-NMR spectroscopy, but it has been difficult to incorporate suitable (19)F-labelled amino acids into synthetic peptides. To avoid the drawbacks of previously used labels, we have rationally designed and synthesized a novel amino acid that suits all theoretical and practical requirements for peptide synthesis and subsequent (19)F-NMR structure analysis [Mikhailiuk et. al, Angew. Chem. 2006, 118, 5787-5789]. The enantiomerically pure L-form of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF(3)-Bpg) carries a CF(3) group that is rigidly attached to the peptide backbone and does not racemize during peptide synthesis. It could be demonstrated for several different peptides that their biological activity is usually not affected by a single label, nor the conformation, as monitored by circular dichroism. Here, we carry out a more detailed structure analysis to evaluate the potential and reliability of CF(3)-Bpg for solid-state NMR, using the well-known alpha-helical antimicrobial peptide PGLa as a test case. We have collected several orientational constraints from the anisotropic (19)F--(19)F dipolar couplings of CF(3)-Bpg in various positions of PGLa embedded in lipid bilayers. These resulting structural parameters are then compared with those previously determined from 4-CF(3)-phenylglycine and 3,3,3-d(3)-alanine labels on the same peptide. The analysis confirms that CF(3)-Bpg does not perturb the alpha-helical conformation of PGLa. Likewise, the helix alignment is shown to follow the established concentration-dependent pattern in realigning from a surface-bound S-state to an obliquely tilted T-state. Hence, the advantages of CF(3)-Bpg over all previously used (19)F-labeled side chains are evident, as they combine ease of chemical incorporation and peptide purification with high NMR sensitivity and absent background signals, allowing a straightforward analysis of the dipolar splittings with no need for chemical shift referencing without any ambiguity in the sign of the couplings.     

Modulation of the antioxidant activity of HO* scavengers by albumin binding: a 19F-NMR study

The interaction between different HO(z.rad;) radical scavengers in a three-component antioxidant system has been investigated by means of 19F-NMR spectroscopy. This system is composed of bovine serum albumin (BSA), trolox, and N-(4-hydroxyphenyl)-trifluoroacetamide (CF(3)PAF). The antioxidant capacity of BSA and trolox has been assessed by measuring the amount of trifluoroacetamide (TFAM) arising from the radical mediated decomposition of CF(3)PAF. When assayed separately, both trolox and BSA behaved as antioxidants, as they were effective to protect CF(3)PAF from HO* radical-mediated decomposition. By contrast, trolox enhanced the production of TFAM in the presence of BSA, thus behaving as a pro-oxidant. Urate, carnosine, glucose, and propylgallate showed antioxidant properties both with or without BSA. CF(3)PAF and trolox were found to bind to BSA with association constants in the order of 5 x 10(3)M(-1) and to compete for the same binding sites. These results have been discussed in terms of BSA-catalysed cross-reactions between trolox-derived secondary radicals and CF(3)PAF.     

Solid-state NMR analysis of the PGLa peptide orientation in DMPC bilayers: structural fidelity of 2H-labels versus high sensitivity of 19F-NMR

The structure and alignment of the amphipathic alpha-helical antimicrobial peptide PGLa in a lipid membrane is determined with high accuracy by solid-state 2H-NMR. Orientational constraints are derived from a series of eight alanine-3,3,3-d3-labeled peptides, in which either a native alanine is nonperturbingly labeled (4x), or a glycine (2x) or isoleucine (2x) is selectively replaced. The concentration dependent realignment of the alpha-helix from the surface-bound "S-state" to a tilted "T-state" by 30 degrees is precisely calculated using the quadrupole splittings of the four nonperturbing labels as constraints. The remaining, potentially perturbing alanine-3,3,3-d3 labels show only minor deviations from the unperturbed peptide structure and help to single out the unique solution. Comparison with previous 19F-NMR constraints from 4-CF3-phenylglycine labels shows that the structure and orientation of the PGLa peptide is not much disturbed even by these bulky nonnatural side chains, which contain CF3 groups that offer a 20-fold better NMR sensitivity than CD3 groups.     

NMR spin trapping: detection of free radical reactions with a new fluorinated DMPO analog

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.     

Incorporation of 19F-substituted aromatic amino acids into membrane proteins from chromatophores of Rhodospirillum rubrum G9+

Chromatophore membranes were isolated from cells of the carotenoidless mutant Rhodospirillum rubrum G9⁺ grown in the presence of several fluorinated aromatic amino acids, solubilized using SDS and the extent of incorporation analyzed using high-resolution ¹⁹F-NMR spectroscopy. 3- and 4-¹⁹F-phenylalanine, 6-¹⁹F-tryptophan and 3-¹⁹F-tyrosine were biosynthetically incorporated into membrane proteins whereas 5-¹⁹F-tryptophan and 2-¹⁹F-phenylalanine were inhibitors of cell growth. The polypeptide chains of the major chromatophore membrane protein the light-harvesting complex, were isolated and shown by high-resolution ¹⁹F-NMR to contain 3-¹⁹F-phenylalanine, which is known to be situated principally within the membrane hydrocarbon layer. Broad-band ¹⁹F-NMR spectra of 3-¹⁹F-phenylalanine-labelled chromatophores showed the phenyl ring to be immobilized within the membrane.     

Cell surface changes during muscle differentiation in vitro: a study with the probe 2,4,6-trinitrobenzene sulphonate.

Cell surface changes during muscle differentiation in vitro, were investigated using the non permeant probe 2,4,6-trinitrobenzene sulphonate (TNBS) in order to label the aminogroups of proteins exposed on the outer surface of the plasma membrane. Surface proteins of chick myotubes and 'mature' unfused myoblasts (myoblasts grown for 7 days in a calcium-depleted medium) were found to bind an equal amount of probe, which is twice the amount bound by surface proteins in 'immature' myoblasts (1--2 days of culture) and fibroblasts. This indicates that a 'remodelling' of the plasma membrane outer surface takes place in the course of muscle cell differentiation even in the absence of cell fusion. Moreover, the total amount of TNBS bound to the surface was 4--5 times greater in myotubes than in unfused myoblasts. This appears to result from the surface expansion which occurs in myotubes during the development of the T tubule system.     

The interaction of trifluoroacetyl peptide chloromethyl ketones with porcine pancreatic elastase. Direct evidence for nonproductive enzyme.inhibitor complexes.

19F NMR investigations of the interactions between elastase and the reversible inhibitors CF3CO-Ala3, CF3CO-Lys-Ala2 and CF3CO-Ala4 show that these peptides have a single mode of binding to the enzyme. Furthermore the results indicate that the CF3CO-group experiences the same environment in all of the reversible complexes formed with these inhibitors. This agrees with the higher affinity of these peptides for the enzyme as compared to the corresponding acetylated inhibitors and confirms our earlier hypothesis of the existence of a specific binding site for the CF3CO-group on the enzyme. The interactions between elastase and the irreversible inhibitors CF3CO-Alan chloromethyl ketone (n = 2, 3, 4, 5) and CF3CO-Lys-Ala4 chloromethyl ketone have been investigated by enzymatic measurements and 19F NMR spectroscopy. The kinetic constants k2 and KI describing the irreversible inhibition are significantly lower for all the CF3CO-peptide chloromethyl ketones with exception of CF3CO-Ala2 chloromethyl ketone, than for the corresponding acetylated ones. Moreover, 19F NMR spectroscopy enabled us to demonstrate, for the tri- and tetrapeptide derivatives, the parallel formation of reversible nonproductive enzyme.inhibitor complexes. The spectroscopic properties of these complexes are completely different from those of the irreversible complexes but are similar to those observed with the reversible complexes described above. In the case of the pentapeptide chloromethyl ketones, fast hydrolysis of the peptide or fast inactivation of the enzyme does not allow observations to be made but does not exclude the existence of reversible nonproductive complexes. In fact, their existence is strongly suggested by the enzyme reaction rate measurements. The similarity of the properties of all the reversible complexes, their striking differences with those of all the irreversible complexes, as well as their mutual exclusivity, permit the conclusion that the CF3CO-group does not bind at one of the classical S subsites of elastase.     

A 19F-NMR study of 2-fluoro-4-demethoxydaunomycin intercalation complexes with the decanucleotides d(G-C)5 and d(A-T)5

Binding configurations and equilibria of intercalation complexes formed by the novel anthracycline drug, 2-fluoro-4-demethoxydaunomycin (2FD), with the decanucleotides d(G-C)5 and d(A-T)5 have been studied by 19F-NMR spectroscopy. The 19F chemical shift of 2FD bound to d(A-T)5 was approximately 1.5 ppm downfield of that observed for 2FD bound to d(G-C)5. By mixing equimolar amounts of aqueous d(G-C)5, d(A-T)5 and 2FD, the distribution of drug between the nucleotides was followed by observing relative peak intensities and showed no G-C or A-T binding preference at room temperature. It was shown that each decanucleotide duplex bound three 2FD molecules, giving a neighbour exclusion parameter, n, of n = 3 for this drug. The stoichiometric complexes, which we denote by [d(A-T)5][2FD]3 and [d(G-C)5][2FD]3, were also purified and isolated in this study.     

A 19F-NMR study of 3-fluoro-4-demethoxydaunomycin intercalation complexes with the hexanucleotide d(CTGCAG)2

Equilibrium systems containing intercalation complexes formed between the novel anthracycline drug, 3-fluoro-4-demethoxydaunomycin (3FD), and the hexanucleotide duplex d(CTGCAG)2 have been studied by 19F-NMR spectroscopy. Solutions containing a 1:1 molar ratio of 3FD/d(CTGCAG)2 gave four 19F signals which have been assigned to each of four possible intercalation isomers for the 1:1 3FD.d(CTGCAG)2 complex, which we denote by [d(CTGCAG)2][3FD]; these were where 3FD bound between the 5'-CT-3', 5'-TG-3', 5'-GC-3' or 5'-CA-3' base sequences, with the drug sugar moiety lying in the minor groove and pointed in the 3' direction in each case. Changes in temperature and NaCl concentration affecting the equilibrium distribution of these isomers were studied and indicated that no overriding binding site preference prevailed under standard biochemical conditions. Formation of some of the 2:1 3FD.d(CTGCAG)2 complex occurred when a solution of [d(CTGCAG)2][3FD] was exposed to excess 3FD; however, this complex was unstable to gel filtration and no co-operative binding of the second 3FD molecule was observed.     

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.     

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.     

19F-nuclear magnetic resonance study of glycerolipid fatty acyl chain order in Acholeplasma laidlawii B membranes

The technique of 19F-nuclear magnetic resonance (19F-NMR) spectroscopy offers a number of advantages for studies of lipid fatty acyl chain orientation and dynamics in biomembranes. However, the geminal difluoromethylene fatty acid probes usually employed in such studies appreciably perturb the organization of lipid bilayers. We have thus synthesized a series of specifically monofluorinated palmitic acids and carried out biophysical, biochemical, and physiological studies establishing their suitability as relatively non-perturbing probes of lipid hydrocarbon chain organization. These 19F-NMR probes were then used to determine the fatty acyl chain order profiles of Acholeplasma laidlawii B membranes highly enriched in a variety of different exogenous fatty acids, particularly those containing a methyl branch or a trans-double bond.     

Application of 19F NMR spectroscopy to a study of carbon monoxide binding to human hemoglobin modified at Cys-beta 93 with the S-trifluoroethyl residue

The S-2,2,2-trifluoroethyl residue (-SCH2CF3) has been incorporated into human hemoglobin, Hb4(SH)2, as a reporter group at Cys-beta 93 using a sequence of disulfide interchange reactions [F. C. Knowles (1981) Anal. Biochem. 110, 19-26]. The 19F NMR spectrum at 235.2 MHz of carboxyhemoglobin (COHb)4(SSCH2CF3)2 was a band 50 Hz wide at half peak height. Conversion of the carbon monoxide derivative to the ligand-free form was accompanied by a downfield shift of 125 Hz (0.53 ppm). Weighed aliquots of solutions of Hb4(SSCH2CF3)2 and (COHb)4(SSCH2CF3)2 were mixed, yielding solutions of known fractional saturation with carbon monoxide. An independent estimate of F of these samples was derived from the amplitudes of the resonance intensities in the 19F NMR spectra. These independent methods for determination of the value of F were not uniformly in agreement. In the presence of inositol hexaphosphate the estimate of F derived from 19F NMR spectra was considerably less than the actual value. The discrepancies between the two independent methods for determining F can be explained by a preferred order of binding of carbon monoxide to the alpha-chains. The preference for binding to the alpha-chains was abolished by removing phosphates. A model for transmission of cooperative effects in hemoglobin was presented which accounted for the characteristic shape of the hemoglobin-oxygen dissociation curves as well as the chain heterogeneity revealed by 19F NMR experiments.     

Redox-sensitive mechanism of no scavenging by nitronyl nitroxides

Nitronyl nitroxides, NN., have been increasingly used in the field of NO-related studies as specific antagonists of NO. . We employed a combination of EPR and NMR spin trapping to study the mechanisms of the reaction of NN. with NO. in reducing environments. EPR allowed observation of NO-induced transformation of the paramagnetic trap, NN., to the corresponding iminonitroxide, IN. . In a complementary way, corresponding EPR-invisible diamagnetic products (the hydroxylamines NN-H and IN-H) were detected by 19F-NMR using newly synthesized fluorinated traps. Addition of reducing agents to a solution of NN. resulted in fast disappearance of its EPR spectra and appearance of a 19F-NMR peak of the corresponding hydroxylamine, NN-H. Addition of NO. as a bolus, or NO. generated on sodium nitroprusside photolysis, resulted in 19F-NMR-detectable accumulation of the hydroxylamine, IN-H. Upon high rates of NO. generation in ascorbate-containing solutions, partial recovery of NN. was observed, which undergoes further reactions with NO. and ascorbate in a competitive manner. Using 19F-NMR and a fluorinated trap, NO-induced conversion of NN-H into IN-H was also observed in vivo in hypertensive ISIAH rats compared with normotensive WAG rats. The results provide insight into a new potential redox-sensitive mechanism of the antagonistic action of NN. against NO., which may provide insight into previously unexplained behavior of this category of NO-reacting compounds.     

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.     

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.     

Measurement of intracellular Ca2+ in young and old human erythrocytes using 19F-NMR spectroscopy.

Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure free ionic intracellular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5'-difluoroBAPTA) and fluorine nuclear magnetic resonance (19F-NMR) techniques to measure changes of intracellular Ca2+ concentrations ([Ca2+]i) with cell aging. We have demonstrated in these studies that human erythrocyte [Ca2+]i is significantly elevated as a function of in-vivo aging. Young cells, the least dense fraction of density-separated erythrocytes, contained an average of 62 (+/- 4) nM Ca2+ (+/- S.E.), whereas the oldest, most dense cell fraction contained 221 nM Ca2+ (+/- 25). Mechanisms by which intracellular [Ca2+] increases with in-vivo aging are currently under investigation.     

Fluorine-NMR studies of chimpanzee hemoglobin

It has been demonstrated that 4-fluorophenylalanine, a known inhibitor of protein synthesis, becomes incorporated into hemoglobin when present in the diet of a chimpanzee. 19F-NMR spectra of various forms of this protein show well-resolved lines, each line presumably corresponding to a unique phenylalanine/fluorophenylalanine position of the primary sequence. Fluorine chemical shifts and, by implication, tertiary structures vary with the oxidation state and ligand.     

Facilitated transport of amino acids across organic phases and the human erythrocyte membrane.

1. An artificial facilitated amino-acid-transfer process operating across a chloroform phase is reported. 2. This process utilizes a family of bis(salicylamidato)copper(II) complexes. 3. A mechanism is proposed for this process and for its sensitivity towards cyanide and bathophenanthroline sulphonate. 4. Facilitated transfer of L-leucine in human erythrocytes has been shown to be inhibited by bathophenanthroline sulphonate.