Proton magnetic relaxation and anion effect in solutions of acid ferricytochrome c.

Measurements of the longitudinal relaxation rates of water protons in aqueous solutions of ferricytochrome c and their temperature dependence, were used for the elucidation of the heme iron ligands at acid pH. The relaxation rates increased with a decrease in pH and pK values of 2.5 and 4.48 were evaluated for the aqueous and 6 m urea solutions, respectively. The results at acid pH are compatible with a structure in which two water molecules exchange rapidly between the coordination sphere of high spin heme iron and the bulk. They suggest that concomitantly with the low-high spin transition the histidine-18 and methionine-80 iron bonds break simultaneously. Addition of various anions, including methanesulfonate at pH 1.95 caused a 85% decrease in the net longitudinal relaxation rate. However, neither the chemical shift nor the width of the methyl proton nmr line of methanesulfonate in solution of acid ferricytochrome c were affected indicating that the effect of anions is not due to a direct binding to the heme iron. The relaxation mechanism of the water molecules in the first coordination sphere of the ferric ion in acid cytochrome c is discussed. It appears that the longitudial relaxation rate is modulated by the electronic correlation time of the ferric ion which was calculated to be τ_s = 6 × 10^?11 sec at 60 MHz.     

Effect of thermal denaturation on water–collagen interactions: NMR relaxation and differential scanning calorimetry analysis

The dependence of the proton spin–lattice relaxation rate, and of the enthalpy and temperature of denaturation on water content, were studied by nmr and differential scanning calorimetry (DSC) in native and denatured collagen. Collagen was first heated at four different temperatures ranging from 40 to 70°C. The percentage of denatured collagen induced by these preheating treatments was determined from DSC measurements. The DSC results are discussed in terms of heat‐induced structural changes. A two‐exponential behavior for the spin–lattice relaxation was observed with the appearance of denatured collagen. This was attributed to the presence of a noncollagen protein fraction. The variations in the different longitudinal relaxation rates as a function of the moisture content and of the denatured collagen percentage are described within the multiphase water proton exchange model. This study highlights the complementarity of the information obtained from the two analytical tools used. © 1999 John Wiley & Sons, Inc. Biopoly 50: 690–696, 1999     

Electron-electron distances in spin-labeled low-spin metmyoglobin variants by relaxation enhancement

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17-30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between ∼30-140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10-30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most variants, distances determined from the temperature dependence of spin-echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II within about an angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement, and the impact diminishes as r⁻³ instead of r⁻⁶, as with spin-lattice relaxation enhancement, which permits measurements at longer distances.     

Relaxation spectra of the iron spin transition in methemoglobin

Temperature-jump relaxation spectra of methemoglobin have been monitored in the spin-sensitive region of the absorption spectrum at pH 6. A single relaxation process is observed, the amplitude of which correlates exactly with that expected for spin state changes. The time-constant is of the order of 1 to 10 ms at 13 °C. Quaternary structural effects perturb the spin dynamics, as evidenced by a slower relaxation in the αβ dimer as opposed to the tetramer. On the other hand, the spin dynamics of the tetramer are not greatly affected by binding saturating amounts of inositol hexaphosphate. This is partly a reflection of the fact that the relative perturbation, caused by inositol hexaphosphate binding, of the equilibrium between high and low-spin species is small, under the conditions studied. In addition, it means that under these conditions, inositol hexaphosphate does not significantly perturb the flexibility of the irons in the heme groups.     

Nmr study of collagen-water interactions

A proton magnetic resonance study of different cross-linked collagens was performed as a function of water content and temperature. Collagens from three connective tissues (calf, steer, and cow) were chosen according to the different number of nonreducible multivalent cross-links, which increases during the life of animal. Samples were hydrated under five well-defined water activities (A_w) ranging from 0.44 to 0.85. The transverse and cross-relaxation times of water protons were studied as a function of temperature from ?20 up to 100°C. From the temperature dependence of relaxation rates, the dynamics of water molecules can be described according to different processes: exchange of protons at the higher temperatures and dipole-dipole interactions that prevail at the lower temperatures. The exchange processes are analyzed as a function of the residence lifetime of water molecules at the protein interface and of the transfer of spin energy from water protons to macromolecule protons. The proton dipole-dipole interactions are related to the relaxation parameters of protein and water protons. All the relaxation parameters showed specific behavior for the 0.44 water activity for every tissue. The collagen tissue from calf also showed distinct behavior in comparison with other tissues. © 1994 John Wiley & Sons, Inc.     

Electron-electron spin-spin interaction in spin-labeled low-spin methemoglobin.

Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1–6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

Spin-dependent absorption of water molecules

The effects of spin state of water molecules on its absorption on lyophilized DNA, lysozyme and some inorganic sorbents were studied. It was shown that the absorption rates of ortho and para water from vapor differ noticeably. The para isomer binding with preparations is distinctly faster than that of the ortho isomer in all cases. Clear-cut distinction in the sorption kinetics is determined by the difference in quantum statistics for spin isomers, which in its turn can give rise to remarkable differences in physico-chemical properties of ortho and para water. This finding opens a wide field of activity in studying fundamental and applied problems relating to the role of the spin state of water molecules in physics, chemistry, biology and medicine.     

Proton magnetic relaxation of manganese (II) tetrakis(4-sulfophenyl)porphine ion in water.

Water proton nuclear magnetic spin-lattice relaxation rates are reported as a function of magnetic field strength for aqueous solutions of manganese tetrakis(4-sulfophenyl)porphine complexes. The manganese(III) complex displays relaxation that is remarkably independent of temperature at low magnetic field and a magnetic field dependence that is characteristic of the electron spin relaxation rates, making a contribution to the correlation time that dominates the electron-nuclear coupling. The manganese(II) complex is much more effective in relaxing water protons, but the usual models of first coordination sphere and outer-sphere relaxation fail to account for the magnitude and the magnetic field dependence of the relaxation rates. The data suggest that the delocalization of the electron density into the ligand system provides an increase in the effectiveness of what may be called the outer-sphere paths for water proton relaxation.     

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.     

On peculiarities of temperature dependences of water spectra in the terahertz frequency domain

We analyzed spectra of light and heavy water at temperatures from 4 up to 50°C in a frequency range of 0.15 to 6.5 THz. It was shown that the amplitude of high-frequency relaxation absorption band with its maximum at 0.5 THz extends with increasing temperature and this temperature dependence for light water has a marked feature at 35–40°C as a sharp growth. This fact is noteworthy because this range corresponds to physiological values of a body temperature of the warm-blooded organisms. At the same time, the analogous temperature dependence for heavy water in the considered temperature range lacks this particular feature. Thus, the water with its properties differs significantly not only from other fluids, but also from its own isotopologues.     

Electron spin relaxation time of Fe(III) in high spin heme proteins.

The electron spin relaxation time of high spin Fe(III), taus, was determined from the frequency dependence (5-100 MHz) of the longitudinal proton relaxation rates of water in solutions of catalase, metmyoglobin and acid ferricytochrome c. In all three high-spin heme proteins the relaxation rates incrased below 25 MHz, while no frequency dependence was observed above that frequency. The results are interpreted by assuming that taus, which modulates the dipolar interaction between the unpaired electrons of the iron and the water protons, is frequently independent. Its value was determined to be (6 +/- 1) - 10(-11) s.     

Effect of Partial H2O-D2O Replacement on the Anisotropy of Transverse Proton Spin Relaxation in Bovine Articular Cartilage

Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the “magic-angle” effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.     

Intervention de l'oxygène atmosphériqoe sur l'absorption d'eau chez Helianthus annuus

The role of atmospheric oxygen on root water absorption in Helianthus annuus . The effect of atmospheric anoxia on root water absorption was studied. The experiments were carried out on intact young sunflowers in controlled temperature, light and gas environment; roots were kept in aerated nutrient solution at constant temperature. The evolution of root water absorption and transpiration rate was measured continuously. Before the experiment, the plant was preconditioned at a high transpiration rate by illumination or by CO₂ free air in darkness. Then the atmospheric oxygen was suppressed for 1 h, after which the normal conditions were restored.
In anoxia and darkness, the root water absorption cannot balance transpiration, so that an important water stress develops in the plant; the light compensates this effect through the photosynthetic oxygen. The supply of oxygen, in darkness or in light, immediately removes inhibition of stomatal closure and of root water absorption. Two mechanisms control water absorption by roots: the fast one occurs in the leaves and the slower one cannot develop without the root system.     

Nuclear magnetic spin-lattice relaxation of water protons caused by metal cage compounds.

The nuclear magnetic spin-lattice relaxation rates of water protons are reported for solutions of manganese(II), copper(II), and chromium(III) cage complexes of the sarcophagine type. As simple aqueous solutions, the complexes are only modest magnetic relaxation agents, presumably because they lack protons on atoms in the first-coordination-sphere protons that are sufficiently labile to mix the large relaxation rate at the metal complex with that of the bulk solvent. The relaxation is approximately modeled using spectral density functions derived for translational diffusion of the interacting dipole moments with the modification that the electron spin relaxation rate is directly included as a contribution to the correlation time. In all cases studied, the electron spin relaxation rate is sufficiently large that it contributes directly to the water-proton spin relaxation process. The poor relaxation efficiency of the cage compound may, however, be improved dramatically by binding the complex to a protein. The efficiency is improved even further if the rotational motion of the protein is reduced drastically by an intermolecular cross-linking reaction. The relaxation efficiency of the cross-linked protein-cage complexes rivals that of the best first-coordination-sphere relaxation agents like [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.