13C and 1H solid state NMR investigation of hydration effects on gluten dynamics

The effect of hydration on the molecular dynamics of soft wheat gluten was investigated by solid state NMR. For this purpose, we recorded static and MAS ¹H spectra and SPE, CP, and other selective ¹³C spectra under MAS and dipolar decoupling conditions on samples of dry and H₂O and D₂O hydrated gluten. Measurements of carbon-proton CP times and several relaxation times (proton T₁, T_1ρ and T₂, and carbon T₁) were also performed. The combination of these techniques allowed both site-specific and domain-averaged motional information to be obtained in different characteristic frequency ranges. Domains with different structural and dynamic behaviour were identified and the changes induced by hydration on the dynamics of different domains could be monitored. The proton spin diffusion process was exploited to get information on the degree of mixing among different gluten domains. The results are consistent with the “loop and train” model proposed for hydrated gluten.     

A solid state NMR study of locust bean gum galactomannan and Konjac glucomannan gels

This paper describes a ¹³C solid state NMR study of hydrated powders and gels of locust bean gum galactomannan-LBG and Konjac glucomannan-KGM. Changes in relative spectral intensities, cross-polarization dynamics (T_CH, T_1ρH) and relaxation times (T_1C, T_1H, T_2H) show that hydration (0–90%) of LBG powders increases the 10⁸ Hz frequency molecular motions, probably reflecting the enhanced motion of non-aggregating segments and chain ends. Slower motions (10⁴–10⁵ Hz) are enhanced only slightly at 90% hydration. LBG gel shows higher spatial distinction between aggregated and non-aggregated segments than the hydrated powder and relaxation times indicate higher mobility for galactose-ramified segments, compared to linear mannose segments. While the dynamics of KGM hydration is similar to that of LBG, i.e. mainly affecting fast 10⁸ Hz motions, the gel is significantly more rigid. Both spectra and relaxation times show that glucose residues in KGM gel are particularly hindered, probably due to their preferential involvement in chain aggregation.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

A pulsed H NMR study of the hydration properties of extruded maize-sucrose mixtures

The molecular mobility in maize and maize-sucrose extudates was studied as a function of the degree of hydration (up to 35% w/w dry basis) using proton spin-spin relaxation NMR. The extrusion of maize systems was found to enhance the molecular mobility of the constituents generating the rigid ‘solid-like’ and the mobile ‘liquid-like’ components of the NMR free induction decay signal.

As the moisture content was increased, the amplitude of the normalised NMR signal arising from the rigid component decreased dramatically for the samples containing sucrose, suggesting an increased mobility and thus a solution type behaviour of the sugar. This hypothesis was supported by the observation of shorter T₂ values for the liquid component of the signal recorded for the samples containing sugar. Sucrose was found to be miscible with the polysaccharide at moisture contents below 15%, but progressively dissolved into the additional water at higher moisture contents. This suggested the occurrence of phase separation over a short distance scale as the water content of the maize-sucrose system was modified. The results are discussed in terms of unequal water partitioning between the maize and the sugar components.     

Using simple 13C NMR linewidth and relaxation measurements to make detailed chemical shift assignments in triacylglycerols and related compounds

Two simple experiments measuring the ¹³C linewidths ν_1/2 and spin–lattice relaxation times T₁ of each of the signals in the spectrum of trilinolein indicate that the ν_1/2 and T₁ values are consistent with the different degrees of motional freedom expected for the various ¹³C nuclei. However, for each chain, the ν_1/2 and T₁ measurements indicate a small reversal in mobility at C-10 relative to C-9 before motional freedom again steadily increases on each chain starting at C-11. The T₁ experiment allows unambiguous assignments of the C-8 signal and C-14 signal, which differ by only 0.010 ppm. Measurements of ¹³C ν_1/2 and T₁ values on tripalmitin provide secure assignments for the C-5 and C-6 signals, for which conflicting assignments have been reported. The T₁ measurements also show that among the tightly clustered C-8 through C-12 signals, the C-11 signals are the most downfield, while the C-12 signals are the most upfield, again contrary to a previous report. Similar measurements of ¹³C ν_1/2 and T₁ values on other triacylglycerols or related compounds may prove equally useful in making chemical shift assignments and detecting any discontinuities in motional freedom along a chain. The benefits and possible limitations of ultrahigh field NMR for studying triacylglycerols and related compounds are discussed.     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

NMR evidence of hydrogen bonding in 1-ethyl-3-methylimidazolium-tetrafluoroborate room temperature ionic liquid

The 1-ethyl-3-methylimidazolium-tetrafluoroborate (EMI–BF₄) room temperature ionic liquid was investigated with NMR techniques. Diffusion coefficients measured at temperatures ranging from 300 to 360 K indicate that phase-change occurred in the vicinity of 333 K, which is supported by ¹¹B quadrupolar relaxation rates. This phase change is ascribed to the transformation of the diffusion particle from ‘discrete ion-pair’ to ‘individual ion’ at temperatures above 335 K due to decomposition of the EMI–BF₄ ion pair. Analysis of the ¹³C dipole–dipole relaxation rates identifies the formation of hydrogen bond (C2HF) between the counterions, EMI⁺ and BF₄ ⁻. This hydrogen bonding may have significant contribution to the higher viscosity of this ionic liquid in comparison with the EMI–AlCl₄ ionic liquid at corresponding temperatures.     

A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva.

Results of a study on the solution behaviour of the cell-wall polysaccharide named ulvan obtained from hot water extraction of a flour of Ulva ‘rigida’ are reported. In particular the spectroscopic properties and ion binding capacity of this charged polysaccharide were studied by circular dichroism and isothermal microcalorimetric titrations in order to gain information on the potential exploitation of this low cost biomass. A marked tendency of this polysaccharide to uptake water was evidenced by studying the proton spin-lattice relaxation times of the solvent, T₁, embedded in this highly charged polysaccharide.     

A C solid state nuclear magnetic resonance spectroscopic study of cork cell wall structure: the effect of suberin removal

Solid state ¹³C NMR measurements of cork, before and after suberin removal, showed that aliphatic suberin is spatially separated from carbohydrate and lignin and experiences higher motional freedom. Two types of chain methylenes, differing in chemical shift and in dynamic properties, were identified in aliphatic suberin. Experimental evidence indicated that the more motionally hindered methylenes are those situated nearer the linkages of aliphatic suberin to the cell wall. These linkages were shown to involve –CH₂O– groups, probably engaged in ester linkages to phenylpropane units and carbohydrate C6 carbons. Spectral intensity changes indicated that, during the first steps of alkaline desuberization, these linkages are broken and the shorter aliphatic suberin chains removed. Longer chains require hydrolysis of the ester linkages within the chains and are removed upon stronger alkaline treatment. T₁(C), T_1ρ(H) and T_1ρ(C) relaxation times have shown that the removal of suberin from cork leads to a motionally restricted and more compact environment, on the megahertz and mid-kilohertz timescales. The properties of cork suberin showed that suberin organization in cork is distinct from that in potato tissue.     

In situ IR and NMR study of the interactions between proton donors and the Re(I) hydride complex [{MeC(CH2PPh2)3}Re (CO)2H]. ReH…H bonding and proton-transfer pathways

The reactions of various proton donors (phenol, hexafluoro-2-propanol, perfluoro-2-methyl-2-propanol, monochloroacetic acid, and tetrafluoroboric acid) with the rhenium (I) hydride complex [(triphos)Re(CO)₂H] (1) have been studied in dichloromethane solution by in situ IR and NMR spectroscopy. The proton donors from [(triphos)Re(CO)₂H…HOR] adducts exhibiting rather strong H…H interactions. The enthalpy variations associated with the formation of the H-bonds (−ΔH = 4.4–6.0 kcal mol⁻¹) have been determined by IR spectroscopy, while the H…H distance in the adduct [(triphos)Re(CO)₂H…HOC(CF₃)₃] (1.83 Å) has been calculated by NMR spectroscopy through the determination of the T_1min relaxation time of the Re---H proton. It has been shown that the [(triphos)Re(CO)₂H…HOR] adducts are in equilibrium with the dihydrogen complex [(triphos)Re(CO)₂(η²-H₂)]⁺, which is thermodynamically more stable than any H-bond adduct.     

NMR relaxation studies of intracellular Na in red blood cells

The state of intracellular Na⁺ in human and dog erythrocytes was characterized by ²³Na-NMR using dysprosium complexes as shift reagents. Intracellular Na⁺ concentrations were determined using integration of the inner Na⁺ NMR signals and measurements of the intracellular volume using ⁵⁹Co-NMR of extracellular Co(CN)³⁻₆. T₂ was found to be significantly shorter than T₁, indicating some binding to macromolecules. While the longitudinal magnetization decay follows a single exponential the transverse magnetization could be fitted with a double-exponential function. It was shown that neither the binding to the inner side of the membrane nor binding to hemoglobin contributes to the relaxation enhancement.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Order and fluidity in the terminal methyl region of dipalmitoyl phosphatidylcholine multibilayers: A comparison of raman and H-NMR spectroscopy

Deuterium magnetic resonance (²H-NMR) and Raman spectroscopy are used to investigate order and fluidity at the terminal methyl position in 16-d₃, 16′-d₃ dipalmitoylphosphatidylcholine (16-d₆ DPPC) multibilayers. These methods reveal substantial motion and disorder in the gel phase, 5–10°C below the gel-liquid crystal phase transition temperature (T_m). The phase transition is sensed in the ²H-NMR spectrum as a reduction in the quadrupole splitting from 14 kHz to 3 kHz. In contrast, the Raman parameter used to characterize the CD₃ vibrations is quite insensitive to the melting process, although an analogous parameter does sense disordering at T_m at the 10 and 10′ position in 10-d₂, 10′-d₂ DPPC. The difference in the response of the NMR and Raman parameters may arise because the vibrational spectrum of the CD₃ group is inhomogenously broadened and is therefore quite sensitive to alterations in the local environment around the methyl group. In contrast, the NMR quadrupole splitting is sensitive to both local motion of the methyl group and, near Tm, to motions of the CD₂ group induced by transgauche isomerizations further up the chain. The difficulties that arise when results from different spectroscopic techniques are compared are demonstrated.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a³⁺₃-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a⁺³₃-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a³⁺₃-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a³⁺₃-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J. Biol. Chem. 259, 15094–15099). The relaxation effect of Cyt a on Cyt a₃ could be measured as the difference in microwave field saturation parameter H_1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T₁ was determined from H_1/2 using the transverse relaxation time T₂. The value of T₂ at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T₁ was related to the Cyt a-Cyt a₃ spin-spin distance utilizing available information on the relative orientation of Cyt a₃-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352–364). By taking into account the relaxation parameters for both g_x and g_z components of the Cyt a₃-azide g-tensor, the angle between the g_z components of the Cyt a and Cyt a₃g-tensors was determined to be between 0 and 18°, and the Cyt a-Cyt a₃ spin-spin distance was found to be 19 ± 8 Å.     

PATHWAYS OF INCORPORATION OF FATTY ACID INTO GLYCEROLIPIDS OF THE MURINE PERIPHERAL NERVOUS SYSTEM IN VIVO: ALTERATIONS IN THE DYSMYELINATING MUTANT TREMBLER MOUSE

In vivo glycerolipid metabolism was studied in sciatic nerves of normal and Trembler mice. The results showed that two kinetically independent pathways were implicated in the labeling of diacylglycerophospholipids from [³H]palmitate: the Kennedy pathway and a ‘direct acylation’ pathway. In normal nerves, 45% of the glycerophospholipids were labeled, with a rate constant k₃ = 3.9 × 10⁻³ min⁻¹, from phosphatidic acid and diacylglycerol intermediates, themselves formed with a rate constant of k₁ = 0.24 min⁻¹ from a free ³H-fatty acid pool, FFA₁, that represents 45% of the total injected label. The remaining 55% of the glycerophospholipids were labeled from a kinetically distinct free ³H-fatty acid pool, FFA₂, with a rate constant of k₄ = 9.8 × 10⁻², via a process that does not implicate a detectably labeled metabolic intermediate (‘direct acylation’). Glycerophospholipid labeling via the Kennedy pathway in the Trembler mouse sciatic nerves was reduced to 75% of the normal level, while labeling via the ‘direct acylation’ pathway was increased 1.4-fold. The values of the rate constants for free ³H-fatty acid utilisation (k₁ and k₄) were both increased about 2.5-fold, while that of glycerophospholipid formation from diacylglycerol (k₃) was close to normal. Copyright © 1996 Elsevier Science Ltd     

C NMR relaxation studies of 1:2 LiCl-ethylaluminum dichloride solutions

A new room-temperature molten salt, 1:2 LiCl-ethylaluminum dichloride (LiCl-EtAlCl₂, f.p. about 178 K), is examined using ¹³C relaxation methods at 7.05 T (−25 to + 80 °C). The methylene carbon undergoes scalar relaxation of the ‘second kind’ as it is coupled to a faster relaxing (quadrupolar) nucleus. LiCl-EtAlCl₂ undergoes a significant liquid-state phase change between 5 and 15 °C as evidenced by observed changes in the relaxation properties of the methylene and methyl carbons and J(¹³C−²⁷Al). The J(¹³C−²⁷Al) coupling constants are 75 (− 10 to + 5 °C) and 11 Hz (15–65 °C), indicating a change in structure between 5 and 15 °C. Chemical shift anisotropies of 56 and 48 ppm are obtained for the methylene and methyl carbons in the EtAlCl₂ dimer part of the 1:2 LiCl-EtAlCl₂ solution.     

Mobility of lipid in complexes of amylose–fatty acids by deuterium and C solid state NMR

Palmitic and lauric acid complexes with amylose were studied by solid state methods: ¹³C CP/MAS NMR, deuterium NMR, X-ray powder diffraction and differential scanning calorimetry (DSC). The crystalline amylose complexes were found to be in a V-type sixfold single chain helix. The melting points of the complexes were over 100°C, at least 40–50°C higher than the melting points of the free fatty acids. CP/MAS ¹³C NMR spectra revealed two resonance peaks at 33.6 and 32.4 ppm for the palmitic acid, which were assigned as free and complexed fatty acid, respectively. A single resonance peak at 32.4 ppm was found for the lauric acid and assigned to the complex. The chemical shift of 32.4 ppm for the complexed fatty acids suggests a combined trans and gauche conformation for the fatty acid chain in the complex. T₁ relaxation measurements on the two palmitic acid resonances show different behavior: a very slow relaxation for the 33.6 ppm and a much faster relaxation (1.2 s) for the 32.4 ppm resonances. The latter was similar to the relaxation of the single resonance of the lauric acid (1.1 s). Temperature dependent deuterium spectra of the amylose–lauric acid and amylose–palmitic acid complexes suggest a complete complexation for the amylose–lauric acid, whereas the amylose–palmitic acid complex is partially disassociated by the thermal treatment. Based on the overall data, a partially disordered model is proposed: an imperfect helix with the fatty acid partly inside and partly out, depending on crystallization conditions and the necessity of placing the carboxyl head outside the V-helix.     

Influence of spent brewer’s yeast β-glucan on gelatinization and retrogradation of rice starch

The effects of β-glucan (BG) prepared from spent brewer’s yeast on gelatinization and retrogradation of rice starch (RS) were investigated as functions of mixing ratio and of storage time. Results of rapid visco-analysis (RVA) indicated that addition of BG increased the peak, breakdown, setback, and final viscosities, but decreased the pasting temperatures of the rice starch/β-glucan (RS/BG) mixtures. Differential scanning calorimetry (DSC) data demonstrated an increase in onset (T_o), peak (T_p), and conclusion (T_c) temperatures and a decrease in gelatinization enthalpy (ΔH₁) with increasing BG concentration. Storage of the mixed gels at 4 °C resulted in a decrease in T_o, T_p, T_c, and melting enthalpy (ΔH₂). The retrogradation ratio (ΔH₂/ΔH₁) and the phase transition temperature range (T_c − T_o) of the mixed gels increased with storage time, but this effect was reduced by the addition of BG. BG addition also slowed the syneresis of the mixed gels. Results of dynamic viscoelasticity measurement indicated that the addition of BG promoted RS retrogradation at the beginning and then retarded it during longer storage times. The added BG also retarded the development of gel hardness during refrigerated storage of the RS/BG mixed gels.     

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.