The state of water in muscle as studied by pulsed NMR

Spin-lattice relaxation times for the water protons in rat gastronemius muscle are reported over the temperature range +37 to −70°C at six resonance frequencies ranging from 4.5 to 60.0 MHz. From −8 to −70°C, the bulk of the muscle water is frozen. The unfrozen part is termed the hydrated layer and amounts to 7–12% of the total water content. Its correlation time takes teh form of a log-Gaussaian distribution function. From +37 to −8°C, the spin-lattice relaxation time is explained by the exchange of water between the hydration layer and the rest of the water, which behaves like ordinary liquid water. The fact that the observed T₂ values are smaller than the calculated values is attributed to the inner field inhomogeneity of the heterogenous system and/or the modification of T₂ due to non-zero dipolar interaction.In the presence of perdeuterated dimethylsulfoxide, the freezing point of water decreases and the amount of non-freezable water increases. T₁ of water protons for muscle containing 10, 20, and 40% dimethylsulfoxide was calculated.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Pulsed NMR studies of water in striated muscle non-freezing factor of water II. Spin-lattice relaxation times and the dynamics of the non-freezing fraction of water

Spin-lattice relaxation times for the water protons in frog gastrocnemius muscle are reported over the temperature range 193 to 283 °K at Larmor frequencies of 30 and 60 MHz. Results of measurements under similar conditions of the transverse relaxation times are also reported. The relaxation times of the non-freezing 20% of the muscle water are interpreted in terms of water molecules, absorbed on or interacting with the proteins, and which are undergoing anisotropic motion, probably with a distribution of correlation times. Proton spin-lattice relaxation times are also reported for muscles under tension, the tension being produced by loading of the muscles with varying weights.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.     

Seasonal changes in the physical state of crown water associated with freezing tolerance in winter wheat

The relationship between freezing tolerance (expressed as LT₅₀, the lethal freezing temperature for 50% of plants) and the amount and physical state (as determined by spin-lattice [T₁] and spin-spin [T₂] relaxation times of protons) of water in crown tissue was examined in contrasting winter wheat (Triticum aestivum L.) varieties grown under field conditions from 1992 to 1994. During acclimation, the LT₅₀ values decreased from around -7 to -17, -20 and -27°C in PI 173438, Chihokukomugi and Valuevskaya, respectively. Tissue water content decreased continuously through autumn to reach a plateau around 3 g H₂O (g dry weight)^-1 in early winter when LT₅₀ was still decteasing, and then gradually increased under snow cover. A significant negative correlation was found between mean minimum air temperatures and freezing tolerance prior to the establishment of continuous snow cover. In contrast, a positive association between mean minimum temperatures and crown tissue water content was significant only when air temperatures were above 0°C, as water content did not decrease further at sub-zero temperatures. Seasonal changes in T₁ were closely related to changes in freezing tolerance. T₁ decreased until January even though water content stopped decreasing. Further tests on 15 field-grown varieties confirmed a strong negative association between freezing tolerance and T₁. The results suggest that cold hardening is comprised of two stages, with the transition occurring at ca 0°C. Development of hardiness was related to (1) a reduction in water content in the first stage (at minimum temperatures > 0°C), and (2) a change in physical state of water without much reduction in water content in the second stage. Varietal differences in hardiness thus arise due to changes in both water content and physical state of water.     

Nuclear magnetic resonance investigation of the state of water in human hair

Measurements have been made of the nuclear magnetic relaxation times T₁ and T₂ of the protons of water in hair. These are interpreted as showing that water molecules in hair exist in a continuous range of environments with a wide spread of rates of molecular rotation. Even at high water contents most of the water molecules are much less mobile than molecules in bulk water. The term “mobility” is given a quantitative meaning.     

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz

TheT ₁ andT ₂ relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency =300 MHz). TheT ₁/T ₂ ratios and the correlation time, τ _c , calculated from theT ₁/T ₂ ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.     

The state of water in biological systems as studied by proton and deuterium relaxation

Careful experiments on the measurement of the intensity of the deuterium NMR signal for ²H₂O in muscle and in its distillate were performed, and they showed that all ²H₂O in muscles is “NMR visible.”The spin-lattice relaxation time (T₁) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to −70°C. T₁ values of deuterons in ²H₂O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to −20°C. Calculations on T₁ for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T₁ values compared to pure water and the frequency dependence of T₁ are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

Water in normal muscle and muscle with a tumor

The total water content, the amount of non-freezable water, and the Na⁺ and K⁺ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T₁) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to −65°C. Quantitatively calculated T₁ values are given. The difference in T₁ for the two types of muscle at temperatures above −5°C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na⁺.     

Nuclear magnetic resonance relaxation times and plasmalemma water exchange in ivy bark

Measurement of nuclear magnetic resonance (NMR) relaxation times (transverse [T₂] and longitudinal [T₁]) for Hedera helix L. cv. Thorndale (ivy) bark water indicates the presence of at least two populations of water with different relaxation characteristics. One population of water with short T₂ and T₁ was found to be composed of both hydration water and extracellular free water. The second population of water with long T₂ and T₁ was identified as intracellular bulk water.     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency     

NMR and DSC Water Study During Osmotic Dehydration of Actinidia deliciosa and Actinidia chinensis Kiwifruit

This study investigated mass transfer and water state changes promoted by osmotic dehydration on two kiwifruit species, Actinidia deliciosa and Actinidia chinensis. Osmotic treatment was performed in a 61.5% w/v sucrose solution at three different temperatures (25, 35 and 45 °C), with treatment time from 0 to 300 min. Treatment time positively influenced kiwifruit water loss and solid gain while temperature significantly affected only water loss. Peleg’s model highlighted that the main response differences between the two species occurred during the initial phase of the osmotic treatment. Thermal properties and relaxation time measurements offered a complementary view concerning the effects of osmotic dehydration on kiwifruit. DSC parameters appeared to be sensitive to water and solid exchange between fruit and osmotic solution. LF-NMR proton T₂ revealed the consequences of the water–solid exchange on the cell compartments, namely vacuole, cytoplasm plus extracellular space and cell wall. During the osmotic treatment, the initial freezing temperature and the freezable water content decrease was dependent on time and treatment temperature, showing a similar tendency for both the kiwifruit species. They evidenced the same treatment response also concerning the reduction of vacuole and the increase of cytoplasm plus extracellular space T₂ values.     

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.     

Probing water compartments and membrane permeability in plant cells by 1H NMR relaxation measurements

¹H NMR relaxation times (T₁ and T₂) in parenchyma tissue of apple can identify three populations of water with different relaxation characteristics. By following the uptake of Mn²⁺ ions in the tissue it is shown that the observed relaxation times originate from particular water compartments: the vacuole, the cytoplasm, and the cell wall/extracellular space.

Proton exchange between these compartments is controlled by the plasmalemma and tonoplast membranes. During the Mn²⁺ penetration experiment, conditions occur that cause the relaxation times of protons of cytoplasmic water to be much shorter than their residence time in the cytoplasm. Then the tonoplast permeability coefficient P_d for water can be calculated from the vacuolar T₁ and T₂ values to be 2.44 10^-5 m·s^-1.

     

Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T ₂ and T ₂*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as “probeless” magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T ₂- and T ₂*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin’s stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T ₂ relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T ₂ relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

1H NMR studies: dynamics of water in gelatin

 Proton magnetic resonance was used to characterize the dynamics of water in gelatin. Both sol and gel states were investigated. Transverse relaxation rates (R ₂) were dependent on the proton frequency measurement. (R ₂) measured with the Carr-Purcell-Meiboom-Gill pulse sequence was dependent on pulse spacing. These observations were interpreted in terms of chemical exchanges between water protons and those of the macromolecules in the sol state, whereas in the gel state the contribution of diffusion through microheterogeneities in the sample seems to provide an additional transverse relaxation mechanism. Received: 10 May 1999 / Revised version: 13 December 1999 / Accepted: 25 January 2000     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Kinetics of the polymerization of hemoglobin S: studies below normal erythrocyte hemoglobin concentration.

The kinetics of polymerization of deoxyhemoglobin S have been studied by measuring transverse water proton relaxation times (T₂) in hemoglobin solutions. As seen by other techniques, the kinetic profile consists of a delay time followed by a decrease in T₂ during polymerization. The length of the delay time can be decreased and the rate of change of T₂ can be increased by increasing the concentration of hemoglobin S or non-gelling hemoglobin or ovalbumin. At a total protein concentration of about 210 mg/ml the kinetic profiles in all three cases are indistinguishable suggesting that a non-specific protein-protein interaction may be involved in the kinetics of polymerization. In addition, it is suggested that no polymer formation occurs during the delay period.