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.     

Spin-Echo Nuclear Magnetic Resonance Evidence for Complexing of Sodium Ions in Muscle, Brain, and Kidney

Na⁺ in muscle, brain, and kidney is shown by spin-echo nuclear magnetic resonance (NMR) to consist of two fractions with different NMR parameters. The slow fraction of Na⁺ in these tissues has NMR relaxation times T₁ and T₂ of 10-15 × 10^-3 sec, which is approximately 4-5 times shorter than for Na⁺ in aqueous NaCl solution. The slow fraction may represent Na⁺ dissolved in structured tissue water. The fast fraction of tissue Na⁺, which is shown to represent approximately 65% of the total tissue Na⁺ concentration, has T₂ less than 1 × 10^-3 sec, which resembles the values of T₂ observed for Na⁺ complexed by synthetic ion-exchange resins. One is drawn to the conclusion that approximately 65% of total Na⁺ in muscle, brain, and kidney is complexed by tissue macromolecules.     

Water Mobility and Distribution in Green Coffee Probed by Time-Domain Nuclear Magnetic Resonance

Water distribution in green coffee was studied by means of pulsed nuclear magnetic resonance (NMR). Hydration experiments for relaxometry measurements were performed by adding either H₂O or D₂O to dried green coffee beans up to 35% (dry basis) or, alternatively, by moisture absorption in a controlled humidity environment. The CPMG experimental relaxation decay curves were acquired using a benchtop time-domain NMR analyzer at each hydration level and as a function of time. All NMR data were fitted according to the Laplace inversion approach to obtain the proton mobility distributions of water in the hydrated beans. By comparing the T ₂ relaxograms of the hydrated beans with the ones observed in the untreated raw beans, it was found that up to ??10% water exhibits a rather restricted proton mobility. Hydration experiments carried out with D₂O highlighted the contribution of the chemical exchange between the water protons and those of the solid matrix to the overall NMR signal. A possible interpretation of the data in terms of the antiplasticizer and plasticizer effect of water is offered.     

Proton relaxation studies in H 2 O-D 2 O mixtures. The binding of manganese (II) to bovine serum albumin

We have used the direct method for determining longitudinal relaxation times of water protons in H₂OD₂O mixtures. The relaxation time of pure water (2.7 sec) increases to 9.0 sec in 10% H₂O-90% D₂O mixture. This larger relaxation time enables us to use the direct method to accurately determine relaxation rate enhancements due to paramagnetic metal ions. The binding parameters for the interaction of manganese(II) to bovine serum albumin determined by this method are in excellent agreement with those determined earlier using pulsed methods.     

Long-lived spin state of a tripeptide in stretched hydrogel

The longitudinal (T ₁), transverse (T ₂), and singlet state (T _s) relaxation times of the geminal backbone protons (CH₂) of l-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D₂O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T ₂ and T _s of the CH₂ protons correspondingly decreased (T _s/T ₂ ~ constant), while the change in T ₁ was relatively small. The largest observed T _s/T ₁ value was 3.3 at 46 % w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D₂O in ²H NMR spectra, and residual dipolar splitting of the CH₂ resonance in ¹H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T ₁, T ₂, and T _s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T ₂ is shorter than T ₁ in the presence of non-zero residual dipolar couplings.     

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.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T₁) and spin-spin (T₂) relaxation times of water in cysts hydrated with D₂O have been measured for hydrations between 1.5 and 0.1 g of D₂O per gram of dry solids. When the relaxation rates (I/T₁, I/T₂) of ²H and ¹⁷O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D₂O (or H₂ ¹⁷O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The ²H and ¹⁷O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.

It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H₂O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T₁ as a function of hydration in the vicinity of 0.6 g H₂O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

     

Oxygen diffusion in phospholipid artificial membranes studied by Fourier transform nuclear magnetic resonance

Spin-lattice (T_i) relaxation mesurements can provide information about the presence of oxygen in the environment of a nucleus, since oxygen, by virtue of its paramagnetic properties, increases T_i relaxation rates. Spin-lattice relaxation times were measured for the choline, fatty acid methylene, and fatty acid methyl protons of sonicated dimyristoyl phosphatidyl choline vesicles in D₂O at several oxygen pressures. The increase in relaxation rate due to oxygen was found to be greater for the fatty acid resonances than for the choline resonance. This was interpreted to indicate the presence of oxygen in the hydrocarbon core of the bilayer. In addition, the T_i relaxation data permitted calculation of the oxygen diffusion coefficient in the water and lipid phases.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

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.     

Nuclear Magnetic Relaxation Study of Tobacco Mosaic Virus Solutions

The molecular order of water in liquid-crystalline 5-28% tobacco mosaic virus (TMV) solutions was studied by proton spin-spin, spin-lattice, and translational self-diffusion coefficient measurements at various concentrations as well as by deuteron D₂O nuclear magnetic resonance (NMR) studies. The results show that the average H₂O molecule spends less than 1% of its time in an ordered state bound to the TMV backbone. The protons on the TMV molecules themselves, on the other hand have a very short spin-spin relaxation time T₂ of about 20 μs, demonstrating the existence of a high degree of liquid-crystalline order.     

Nuclear magnetic resonanceimaging of membrane permeability changes in plants during osmoticstress

The cell water balance of maize (Zea mays L., cv LG 11) andpearl millet (Pennisetum americanum L., cv MH 179) duringosmotic stress was studied non‐invasively using ¹H nuclearmagnetic resonance (NMR) microscopy. Single NMR parameter imagesof (i) the water content (ii) the transverse relaxation time (T₂)and (iii) the apparent diffusion coefficient (D_app)were used to follow the water status of the stem apical region duringosmotic stress. During stress there are hardly any changes in watercontent or T₂ of the stem region of maize. Incontrast, the apical tissue of pearl millet showed a ～ 30% decreaseof T₂ within 48 h of stress, whereasthe water content and D_app did not change. Thesechanges can be explained by an increase of the membrane permeabilityfor water. This conclusion is supported by results from scanningelectron microscopy, relaxation measurements of sugar solutionsand numerical simulations of the relaxation and (apparent) diffusionbehaviour of water in a plant cell.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

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.     

Pulsed NMR studies on water in striated muscle III. The effects of water content

Pulsed NMR is used to study the kinetics of dehydration of frog gastrocnemius muscle. In addition, measurements are reported of the variation of the spin-lattice (T₁) and transverse (T₂) nuclear magnetic relaxation times of the water protons as a function of water content. The proton transverse relaxation and freezing properties of the water in muscles which had been dehydrated and then rehydrated are also investigated. Correlation of the double-exponential dehydration kinetics with the transverse relaxation at various water contents provides strong evidence for the evidence of a fraction of muscle water (10–20%) which is sufficiently strongly held to the solid substance of the muscle to make it relatively slowly removed under conditions of zero relative humidity but which is still dynamically very mobile on average. This is supported by the dependence of T₁ on water content. The relaxation times are interpreted qualitatively in terms of a number of possible effects which are at present not distinguishable. The properties of the dehydrated-rehydrated muscles indicated changes in the muscle proteins which affect the transverse relaxation of the water protons and the freezing properties of the muscle water.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

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.