Detection and characterization of boric acid and borate ion binding to cytochrome c using multiple quantum filtered NMR

The application of multiple quantum filtered (MQF) NMR to the identification and characterization of the binding of ligands containing quadrupolar nuclei to proteins is demonstrated. Using relaxation times measured by MQF NMR multiple binding of boric acid and borate ion to ferri and ferrocytochrome c was detected. Borate ion was found to have two different binding sites. One of them was in slow exchange, k(diss) = 20 +/- 3 s(-1) at 5 degrees C and D(2)O solution, in agreement with previous findings by (1)H NMR (G. Taler et al., 1998, Inorg. Chim. Acta 273, 388-392). The triple quantum relaxation of the borate in this site was found to be governed by dipolar interaction corresponding to an average B-H distance of 2.06 +/- 0.07 A. Other, fast exchanging sites for borate and boric acid could be detected only by MQF NMR. The binding equilibrium constants at these sites at pH 9.7 were found to be 1800 +/- 200 M(-1) and 2.6 +/- 1.5 M(-1) for the borate ion and boric acid, respectively. Thus, detection of binding by MQF NMR proved to be sensitive to fast exchanging ligands as well as to very weak binding that could not be detected using conventional methods.     

Isotope Effects on theO,H Coupling Constant and theO–{H} Nuclear Overhauser Effect in Water

The NMR spectra of solutions of 30%¹⁷O-enriched H₂O and D₂O in nitromethane display the resonances of the three isotopomers H₂O, HDO, and D₂O. All¹⁷O,¹H and¹⁷O,²H coupling constants and the primary and secondary isotope effects onJ(¹⁷O,¹H) have been determined. The primary effect is −1.0 ± 0.2 Hz and the secondary effect is −0.07 ± 0.04 Hz. Using integrated intensities in the¹⁷O NMR spectra, the equilibrium constant for the reaction H₂O + D₂O 2HDO is found to be 3.68 ± 0.2 at 343 K. From the relative integrated intensities of proton-coupled and -decoupled spectra the¹⁷O–{¹H} NOE is estimated for the first time, resulting in values of 0.908 and 0.945 for H₂O and HDO, respectively. This means that dipole–dipole interactions contribute about 2.5% to the overall¹⁷O relaxation rate in H₂O dissolved in nitromethane.     

Al Chemical Shielding Anisotropy

Accurate values for the²⁷Al chemical shielding anisotropy (CSA) are reported for sapphire (α-Al₂O₃). The values (δ_σ= −17.3 ± 0.6 ppm, η_σ= 0.03 ± 0.06) are obtained from single-crystal²⁷Al NMR and appear to be the first convincing determination of an²⁷Al CSA.     

Determination of the (Na) Sternheimer antishielding factor by Na NMR spectroscopy on sodium oxide chloride, Na3OCl

The (Na⁺) Sternheimer antishielding factor γ_∞ (Na⁺) was determined by ²³Na NMR spectroscopy on sodium oxide chloride, Na₃OCl. The quadrupolar coupling constant of the sodium ion in Na₃OCl was determined to QCC=11.34 MHz, which presents the largest coupling constant of a sodium nucleus observed so far. Applying a simple point charge model, the largest principal value of the electric field gradient at the sodium site was calculated to V_zz=−6.76762·10²⁰ V/m². From these values we calculated the (Na⁺) Sternheimer antishielding factor to γ_∞ (Na⁺)=−5.36. In sodium oxide, Na₂O, we observed an isotropic chemical shift of δ_CS=55.1 ppm, referenced to 1 M aqueous NaCl (δ=0 ppm).     

Quantitative Evaluation of the Lactate Signal Loss and Its Spatial Dependence in PRESS Localized H NMR Spectroscopy

Localized ¹H NMR spectroscopy using the 90°−t₁−180°−t₁+t₂−180°−t₂−Acq. PRESS sequence can lead to a signal loss for the lactate doublet compared with signals from uncoupled nuclei which is dependent on the choice of t₁ and t₂. The most striking signal loss of up to 78% of the total signal occurs with the symmetrical PRESS sequence (t₁=t₂) at an echo time of 2/J (290 ms). Calculations have shown that this signal loss is related to the pulse angle distributions produced by the two refocusing pulses which leads to the creation of single quantum polarization transfer (PT) as well as to not directly observable states (NDOS) of the lactate AX₃ spin system: zero- and multiple-quantum coherences, and longitudinal spin orders. In addition, the chemical shift dependent voxel displacement (VOD) leads to further signal loss. By calculating the density operator for various of the echo times TE=n/J, n=1, 2, 3, …, we calculated quantitatively the contributions of these effects to the signal loss as well as their spatial distribution. A maximum signal loss of 75% can be expected from theory for the symmetrical PRESS sequence and TE=2/J for Hamming filtered sinc pulses, whereby 47% are due to the creation of NDOS and up to 28% arise from PT. Taking also the VOD effect into account (2 mT/m slice selection gradients, 20-mm slices) leads to 54% signal loss from NDOS and up to 24% from PT, leading to a maximum signal loss of 78%. Using RE-BURP pulses with their more rectangular pulse angle distributions reduces the maximum signal loss to 44%. Experiments at 1.5 T using a lactate solution demonstrated a maximum lactate signal loss for sinc pulses of 82% (52% NDOS, 30% PT) at TE=290 ms using the symmetrical PRESS sequence. The great signal loss and its spatial distribution is of importance for investigations using a symmetrical PRESS sequence at TE=2/J.     

Al and Si MAS NMR investigations of cordierite glass, μ- and α-cordierite

²⁷Al and ²⁹Si Magic-Angle Spinning NMR results are reported for conventionally prepared glass of cordierite stoichiometry (2MgO · 2Al₂O₃ · 5SiO₂), the metastable high-quartz solid solution (μ-cordierite) and the high-temperature polymorph of cordierite (α-cordierite). Both, ²⁷Al two-dimensional (2D) quadrupole nutation experiments and ²⁷Al satellite transition spectroscopy (SATRAS) have been applied to identify two different tetrahedrally-coordinated aluminium sites (AlO₄). SATRAS has been used to extract the quadrupole interaction parameters and their distribution, the isotropic chemical shifts and the relative populations of the different Al sites. Both, the ²⁷Al and ²⁹Si NMR results, lead to the conclusion that a perfect Si/Al disorder does not exist in these investigated cordierite samples.     

A Study of Dipolar Interactions and Dynamic Processes of Water Molecules in Tendon byH andH Homonuclear and Heteronuclear Multiple-Quantum-Filtered NMR Spectroscopy

The effect of proton exchange on the measurement of¹H–¹H,¹H–²H, and²H–²H residual dipolar interactions in water molecules in bovine Achilles tendons was investigated using double-quantum-filtered (DQF) NMR and new pulse sequences based on heteronuclear and homonuclear multiple-quantum filtering (MQF). Derivation of theoretical expressions for these techniques allowed evaluation of the¹H–¹H and¹H–²H residual dipolar interactions and the proton exchange rate at a temperature of 24°C and above, where no dipolar splitting is evident. The values obtained for these parameters at 24°C were 300 and 50 Hz and 3000 s⁻¹, respectively. The results for the residual dipolar interactions were verified by repeating the above measurements at a temperature of 1.5°C, where the spectra of the H₂O molecules were well resolved, so that the¹H–¹H dipolar interaction could be determined directly from the observed splitting. Analysis of the MQF experiments at 1.5°C, where the proton exchange was in the intermediate regime for the¹H–²H dipolar interaction, confirmed the result obtained at 24°C for this interaction. A strong dependence of the intensities of the MQF signals on the proton exchange rate, in the intermediate and the fast exchange regimes, was observed and theoretically interpreted. This leads to the conclusion that the MQF techniques are mostly useful for tissues where the residual dipolar interaction is not significantly smaller than the proton exchange rate. Dependence of the relaxation times and signal intensities of the MQF experiments on the orientation of the tendon with respect to the magnetic field was observed and analyzed. One of the results of the theoretical analysis is that, in the fast exchange regime, the signal decay rates in the MQF experiments as well as in the spin echo or CPMG pulse sequences (T₂) depend on the orientation as the square of the second-rank Legendre polynomial.     

Isospin properties of () reactions for the formation of deeply-bound antikaonic nuclei

The formation of deeply-bound antikaonic nuclear states in nuclear (K⁻,N) reactions is investigated theoretically within a distorted-wave impulse approximation (DWIA), considering the isospin properties of the Fermi-averaged elementary amplitudes. We calculate the formation cross sections of the deeply-bound states by the (K⁻,N) reactions on the nuclear targets, ¹²C and ²⁸Si, at incident K⁻ lab momentum p_K⁻=1.0 GeV/c and θ_lab=0°, introducing a complex effective nucleon number N_eff for unstable bound states in the DWIA. The results show that the deeply-bound states can be populated dominantly by the (K⁻,n) reaction via the total isoscalar ΔT=0 transition owing to the isospin nature of the amplitudes, and that the cross sections described by ReN_eff and ArgN_eff enable to deduce the structure of the nuclear states; the calculated inclusive nucleon spectra for a deep -nucleus potential do not show distinct peak structure in the bound region. The few-body and states formed in (K⁻,N) reactions on s-shell nuclear targets, ³He, ³H and ⁴He, are also discussed.     

Correlation between P chemical shift tensor and local structure in lithium cyclohexaphosphates Li6P6O18·3H2O and Li6P6O18

To understand the surprising behavior between the variations of the P′–P–P″ angles and the correlated variations of the O′–P–O″ ones, two lithium cyclohexaphosphate compounds Li₆P₆O₁₈·3H₂O and Li₆P₆O₁₈ are studied by solid state nuclear magnetic resonance (NMR) spectroscopy. The two compounds exhibit the same [P₆O₁₈]⁶⁻ ring anions but with 3m or internal symmetry, respectively. Such symmetries induce local distortions that are exhibited by NMR spectroscopy. One-dimensional (1D) NMR gives information on structural sites of ⁷Li and ³¹P ions and the crystallographic non-equivalencies are observed. Nevertheless, in the anhydrous compound, X-ray diffraction and NMR results do not completely agree and some discrepancy exists between the number of sites observed with the first technique and the number of lines exhibited in the NMR spectra either for ⁷Li or ³¹P nuclei. This problem is elucidated by using 2D double quantum NMR spectroscopy coupled with theoretical considerations. We find that the ³¹P chemical shift tensor is dependent on the deviations of the O–P–O angles from those in the regular tetrahedron. Within the same empirical model, we suggest that the surprising behavior between the variations of the P′–P–P″ and the ones of the O′–P–O″ is related to the overall charge on the PO₄ group. We also find the positions of the isotropic lines for ⁷Li essentially depend on the site co-ordination of this nuclei.     

Li mobility in Li0.5 − xNaxLa0.5TiO3 perovskites (0 ≤ x ≤ 0.5): Influence of structural and compositional parameters

The dependence of Li mobility on structure and composition of Li_0.5 − xNa_xLa_0.5TiO₃ perovskites (0 ≤ x ≤  0.5) has been investigated by means of neutron diffraction, nuclear magnetic resonance and impedance spectroscopy. At 300 K, all samples display a rhombohedral superstructure (R-3c S.G.), where octahedra are out of phase tilted along [111] direction of the ideal cubic cell. The elimination of the octahedral tilting is responsible for the rhombohedral–cubic transformation, detected near 1000 K. In these perovskites, La and Na cations are randomly distributed in A sites, but Li ions are fourfold coordinated at unit cell faces of the cubic perovskite. Lithium conductivity, σ_300 K, decreases with the sodium content, decreasing from values typical of fast ionic conductors, 10^− 3 S/cm, to those of good insulators, 10^− 10 S/cm, when the interconnectivity between vacant A sites is lost (x > 0.3). In samples with x < 0.3, dc conductivity displays a non-Arrhenius behaviour, decreasing activation energy from ~ 0.37 to 0.25 eV when the sample is heated between 77 and 500 K. The temperature dependence of B_Li factors shows the existence of two regimes for Li motion. Below 373 K, Li ions remain partially located near square oxygen windows that connect contiguous A sites, but above 400 K, extended Li motions become dominant. The additional decrease of activation energy from 0.25 to 0.16 eV (low-temperature ⁷Li NMR value), should require the full elimination of octahedral tilting which is only produced above 1000 °C.     

Al and Cu NMR study in UCu5Al

We have studied the microscopic properties of the tetragonal UCu₅Al Kondo compound by ²⁷Al and ^63,65Cu NMR in the paramagnetic state. NMR and susceptibility measurements performed on the powdered sample, but oriented along the applied field, showed χ>χ. Plots of K(T) against χ(T) at temperatures T≥100 K yield the transferred hyperfine fields of +5.9 kOe/μ_B for ²⁷Al nuclei, and +5.3 and −7.0 kOe/μ_B for ⁶⁵Cu nuclei in crystallographically inequivalent Cu(2) and Cu(1) sites, respectively. The Knight shift vs. susceptibility plots for T<100 K exhibit a deviation from the linear behaviour (absolute values of shifts become smaller than expected). We attribute this finding to the crystalline electric field effect in similar way as it was reported for several Ce-based compounds. The random distribution of the Al and Cu(2) atoms in the crystal lattice we consider as a reason of an unusual broadening of the NMR spectra, particularly at low temperatures.     

Preparation and electrical properties of a pyrochlore-related Ce2Zr2O8−x phase

A pyrochlore-related Ce₂Zr₂O_8−x phase has been prepared in a reduction reoxidation process from Ce_0.5Zr_0.5O₂ powders. Ce₂Zr₂O_8−x, based on a cubic symmetry with a=1.053 nm, decomposes in nitrogen at 800 °C, but remains stable up to 900 °C in air. It shows mixed oxygen ionic and electronic conductivity. The bulk conductivity at 700 °C is 4×10⁻⁴ S cm⁻¹ in air and 1×10⁻² S cm⁻¹ in nitrogen, and the activation energy is 1.27 eV in air. In nitrogen, the Arrhenius law is not obeyed, and a curved plot was obtained from 400 to 700 °C; then, the conductivity decreased rapidly due to the thermal decomposition of Ce₂Zr₂O_8−x.     

Electrical and mechanical properties of proton conducting SrCe0.95Yb0.05O3 − α

Yb³⁺-doped ceramic strontium cerate of exactly the composition SrCe_0.95Yb_0.05O_{3 − α} was prepared, having a relative density of 99.0 (± 0.3%). Great care was taken to obtain homogeneous, carbonate free material. Analysis are made of the X-ray powder diffraction pattern of the as-prepared dense ceramic, resulting in the orthorhombic unit cell parameters a = 6.997(2) Å, b = 12.296(3) Å, c = 8.588(2) Å, Z = 8 and d_x = 5.806(2) g cm⁻³. Bending strength values of the ceramic in non-proton and proton conducting state are found to be 177 and 194 MPa respectively. The ceramic kept under proton conducting conditions for 500 h at 300 °C to 800 °C in a N₂ flow containing 155 mbar water vapour and 245 mbar H₂, have shown to remain chemically and structurally stable. Impedance spectroscopy measurements of the bulk conductivity of the proton conducting ceramic revealed an activation energy of 53.2 kJ mol⁻¹ and a preexponential factor of 359.1 (Ω cm)⁻¹ K. In the non-proton conducting state the ceramic is mainly oxygen ion vacancy conducting, which indicates that charge compensation on substituting Yb⁺³ in SrCeO₃ takes place by oxygen ion vacancies.     

One- and two-dimensional C–H/N–H dipolar correlation experiments under fast magic-angle spinning for determining the peptide dihedral angle φ

A recently proposed ¹³C–¹H recoupling sequence operative under fast magic-angle spinning (MAS) [K. Takegoshi, T. Terao, Solid State Nucl. Magn. Reson. 13 (1999) 203–212.] is applied to observe ¹³C–¹H and ¹⁵N–¹H dipolar powder patterns in the ¹H–¹⁵N–¹³C–¹H system of a peptide bond. Both patterns are correlated by ¹⁵N-to-¹³C cross polarization to observe one- or two-dimensional (1D or 2D) correlation spectra, which can be simulated by using a simple analytical expression to determine the H–N–C–H dihedral angle. The 1D and 2D experiments were applied to N-acetyl[1,2-¹³C,¹⁵N] -valine, and the peptide φ angle was determined with high precision by the 2D experiment to be ±155.0°±1.2°. The positive one is in good agreement with the X-ray value of 154°±5°. The 1D experiment provided the value of φ=±156.0°±0.8°.     

Molecular dynamics and information on possible sites of interaction of intramyocellular metabolites in vivo from resolved dipolar couplings in localized H NMR spectra

Proton NMR resonances of the endogenous metabolites creatine and phosphocreatine ((P)Cr), taurine (Tau), and carnosine (Cs, β-alanyl-l-histidine) were studied with regard to residual dipolar couplings and molecular mobility. We present an analysis of the direct ¹H–¹H interaction that provides information on motional reorientation of subgroups in these molecules in vivo. For this purpose, localized ¹H NMR experiments were performed on m. gastrocnemius of healthy volunteers using a 1.5-T clinical whole-body MR scanner. We evaluated the observable dipolar coupling strength SD₀ (S = order parameter) of the (P)Cr-methyl triplet and the Tau-methylene doublet by means of the apparent line splitting. These were compared to the dipolar coupling strength of the (P)Cr-methylene doublet. In contrast to the aliphatic protons of (P)Cr and Tau, the aromatic H2 (δ = 8 ppm) and H4 (δ = 7 ppm) protons of the imidazole ring of Cs exhibit second-order spectra at 1.5 T. This effect is the consequence of incomplete transition from Zeeman to Paschen-Back regime and allows a determination of SD₀ from H2 and H4 of Cs as an alternative to evaluating the multiplet splitting which can be measured directly in high-resolution ¹H NMR spectra. Experimental data showed striking differences in the mobility of the metabolites when the dipolar coupling constant D₀ (calculated with the internuclear distance known from molecular geometry in the case of complete absence of molecular dynamics and motion) is used for comparison. The aliphatic signals involve very small order parameters S ≈ (1.4 − 3) × 10⁻⁴ indicating rapid reorientation of the corresponding subgroups in these metabolites. In contrast, analysis of the Cs resonances yielded S ≈ (113 − 137) × 10⁻⁴. Thus, the immobilization of the Cs imidazole ring owing to an anisotropic cellular substructure in human m. gastrocnemius is much more effective than for (P)Cr and Tau subgroups. Furthermore, ¹H NMR experiments on aqueous model solutions of histidine and N-acetyl-l-aspartate (NAA) enabled the assignment of an additional signal component at δ = 8 ppm of Cs in vivo to the amide group at the peptide bond. The visibility of this proton could result from hydrogen bonding which would agree with the anticipated stronger motional restriction of Cs. Referring to the observation that all dipolar-coupled multiplets resolved in localized in vivo ¹H NMR spectra of human m. gastrocnemius collapse simultaneously when the fibre structure is tilted towards the magic angle (θ ≈ 55°), a common model for molecular confinement in muscle tissue is proposed on the basis of an interaction of the studied metabolites with myocellular membrane phospholipids.     

() reaction studies of Dy and Hf

Angular distributions for the ¹⁶³Dy(t,p) and ¹⁷⁷Hf(t,p) reactions were measured using 17 MeV tritons from the McMaster University Tandem Van de Graaff accelerator. Reaction products were analyzed with a magnetic spectrograph and detected with photographic emulsions. Favored L=0 transitions confirmed assignments of the 5/2⁻[523] band in ¹⁶⁵Dy and 7/2⁻[514] band in ¹⁷⁹Hf. Additional L=0 transitions in each nuclide identified previously unknown 5/2⁻ levels in ¹⁶⁵Dy and 7/2⁻ ones in ¹⁷⁹Hf. Overall trends of L=0 strengths support the existence of subshell closures at neutron numbers 98 and 108. On the basis of a relatively strong L=2 transition, the K^π=11/2⁻ γ-vibration based on the 7/2⁻[514] state is identified at 1689 keV in ¹⁷⁹Hf, about 440 keV above its previously-assigned K^π=3/2⁻ partner.     

In vivo NMR T2 relaxation of experimental brain tumors in the cat: A multiparameter tissue characterization

Experimental gliomas (F98) were inoculated in cat brain for the systematic study of their in vivo T₂ relaxation time behavior. With a CPMG multi-echo imaging sequence, a train of 16 echoes was evaluated to obtain the transverse relaxation time and the magnetization M(0) at time t = 0. The magnetization decay curves were analyzed for biexponentiality. All tissues showed monoexponential T₂, only that of the ventricular fluid and part of the vital tumor tissue were biexponential. Based on these NMR relaxation parameters the tissues were characterized, their correct assignment being assured by comparison with histological slices. T₂ of normal grey and white matter was 74 ± 6 and 72 ± 6 msec, respectively. These two tissue types were distinguished through M(0) which for white matter was only 0.88 of the intensity of grey matter in full agreement with water content, determined from tissue specimens. At the time of maximal tumor growth and edema spread a tissue differentiation was possible in NMR relaxation parameter images. Separation of the three tissue groups of normal tissue, tumor and edema was based on T₂ with T_2(normal) < T_2(tumor) < T_2(edema). Using M(0) as a second parameter the differentiation was supported, in particular between white matter and tumor or edema. Animals were studied at 1–4 wk after tumor implantation to study tumor development. The magnetization M(0) of both tumor and peritumoral edema went through a maximum between the second and third week of tumor growth. T₂ of edema was maximal at the same time with 133 ± 4 msec, while the relaxation time of tumor continued to increase during the whole growth period, reaching values of 114 ± 12 msec at the fourth week. Thus, a complete characterization of pathological tissues with NMR relaxometry must include a detailed study of the developmental changes of these tissues to assure correct experimental conditions for the goal of optimal contrast between normal and pathological regions in the NMR images.     

Analogous study in low magnetic field between Cu–Ge and Cu–Si ferrites

Two series of mixed copper ferrites, Cu_1+x Ge_x Fe_2−2x O₄ and Cu_1+x Si_x Fe_2−2x O₄, have been analogously investigated for x=0.0, 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3. The two systems were prepared using the standard ceramic techniques. X-ray diffraction analysis indicates that both systems formed in a single phase cubic spinel structure. The lattice parameter has a constant value (0.838 nm±0.001) for the two series. The grain diameter was estimated from the scanning electron microscope micrographs for the two series. Some magnetic properties were measured at room temperature. The magnetization M was measured in the range of magnetizing field up to 5500 Am⁻¹. The relative permeability (μ_r) was calculated from the B–H relation. The B–H loops were measured at constant magnetizing current (I=2.5 A which is equivalent to 900 Am⁻¹). Also, the hystersis area and the magnetic parameters B_r, B_s, m_R (B_r/B_s) and apparent energy loss (E) were estimated from the B–H loops; μ_r, B_r, B_s and E are composition dependent.     

Control of susceptibility-related image contrast by spin-lock techniques

Macroscopic magnetic field inhomogeneities might lead to image distortions, while microscopic field inhomogeneities, due to susceptibility changes in tissues, cause spin dephasing and decreasing T₂ relaxation time. The latter effects are especially observed in the trabecular bone and in regions adjacent to air-containing cavities when gradient-echo sequences are applied. In conventional MRI, these susceptibility-related signal voids can be avoided by applying spin-echo (SE) techniques. In this study, an alternative method for the examination and control of susceptibility-related effects by spin-lock (SL) radiofrequency pulses is presented: SL pulses were applied in two different susceptibility-sensitive sequence types: (a) between the jump and return 90° pulses in a 90°_x−τ−90°_−x magnetization-prepared Fast Low Angle Shot (FLASH) sequence and (b) between the 90° pulse and the 180° pulse in an asymmetric SE sequence. The range of Larmor frequencies used for spin locking can be determined for different B₁ amplitudes of the SL pulses, allowing control of image contrast by the amplitude of the SL pulses.     

3D 1H-13C-14N correlation solid-state NMR spectrum

Nitrogen-14 (spin I = 1) has always been a nucleus difficult to observe in solid-state NMR and until recently its observation was restricted to one-dimensional (1D) spectra. We present here the first 3D ¹H–¹³C–¹⁴N NMR correlation spectrum. This spectrum was acquired on a test sample l-histidine·HCl·H₂O using a recently developed technique, which consists in indirectly observing ¹⁴N nuclei via dipolar recoupling with an HMQC-type experiment.