Mobility of water bound to biological membranes: A proton NMR relaxation study

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10^?9 cm²/s.     

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA

The results of semiselective ¹H-nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D₂O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A-T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s^?1 at 25°C. Corresponding values for the G-C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A-T and G-C imino protons, resembling the effect recently observed for poly(rA)-poly(rU) and poly(rI)-poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base-pair opening rates as assumed in the past. Lower limits to the base-pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 10³ s^?1 for the A-T, and 50 s^?1 for the G-C, base paris at 62°C.     

Viscosity of water in hibernating and nonhibernating mammals estimated by proton NMR relaxation times.

Longitudinal (T1) and transverse (T2) nuclear magnetic resonance relaxation times were measured in vitro at 37, 30, 25, 15, and 5 degrees C on serum, brain, liver, kidney, and heart samples from a hibernator, the European hamster, active in summer (SA), active in winter, or in the hibernating state in winter; from a less efficient hibernator, the golden hamster; and from a homeotherm, the rat. T1 and T2 relaxation times varied between species and in the European hamster between the active and hibernating subjects. Despite the major relaxation time differences between the organs, NMR relaxation time measurements showed a general trend to an increase in the viscosity of water for the European hamster in the active state. Although these modifications were not directly related to the process of hibernation itself, the relaxation times observed in the hibernating animals were closer to those seen in the rat. This evidenced that changes of physical properties of water reflect a better adaptation to low temperatures of the hamster, as compared to the nonhibernator, given that the low water viscosity of SA hamster allows the decrease of the viscosity with temperature during the hibernating state. These in vitro studies permit the study the viscosity which is an important physicochemical parameter involved in NMR longitudinal relaxation time of water proton. More detailed studies of other physiological parameters must be undertaken by further in vivo measurements.     

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 2-H2 O in muscle and in its distillate were performed, and they showed that all 2-H2 O muscle 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 degrees C. T1 values of deuterons in 2H2 O 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 degrees 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.     

A proton magnetic relaxation study of ferric myoglobin and haemoglobin in water/ethanediol solutions.

Structural alterations of the haem vicinity of the high-spin derivatives of bovine ferric myoglobin (metmyoglobin) and human haemoglobin and the changes of the interaction with inositol hexaphosphate induced by ethanediol were monitored by solvent-proton magnetic relaxation. On addition of ethanediol up to 60% the fluoromet derivatives exhibit a gradual increase in the accessibility of the haem for the molecules from the solvent. In aquomethaemoglobin solutions with more than 25% ethanediol there is no unique explanation of proton magnetic relaxation. Ethanediol enhances the binding of inositol hexaphosphate to methaemoglobin, but the structural consequences of this binding on the haem-pockets seem to be diminished. The mechanisms of the observed structural and functional alterations of myoglobin as well as haemoglobin tetramer are discussed here.     

Fitting fluorescence emission spectra of probes bound to biological membranes

We show that fluorescence emission spectra for molecules containing the dansyl fluorophor can be accurately described as skewed Gaussians, and that spectra for dansyl probes bound to biological membranes can be resolved using least-squares techniques into two components, representing probe bound to the lipid and protein sites in the membrane.     

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.     

Phosphorus-31 NMR relaxation studies of diethyl P-methoxyphenyl phosphate bound to phosphotriesterase

The effect of Mn²⁺/Mn²⁺, Mn²⁺/Zn²⁺ and Mn²⁺/Cd²⁺ reconstituted phosphotriesterase on the ³¹P spin lattice (1/T₁) relaxation rate of diethyl p-methoxyphenyl phosphate has been investigated. In the presence of Mn²⁺/Mn²⁺ phosphotriesterase, the spin lattice relaxation rate of the phosphorus atom is enhanced giving an upper limit for the phosphorus-metal root mean-sixth average distance of 4.2 Å. These results demonstrate for the first time that substrates for phosphotriesterase bind in close proximity to the binuclear metal center.     

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.     

Structured water layers adjacent to biological membranes

Water amid the restricted space of crowded biological macromolecules and at membrane interfaces is essential for cell function, though the structure and function of this "biological water" itself remains poorly defined. The force required to remove strongly bound water is referred to as the hydration force and due to its widespread importance, it has been studied in numerous systems. Here, by using a highly sensitive dynamic atomic force microscope technique in conjunction with a carbon nanotube probe, we reveal a hydration force with an oscillatory profile that reflects the removal of up to five structured water layers from between the probe and biological membrane surface. Further, we find that the hydration force can be modified by changing the membrane fluidity. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine gel (Lbeta) phase bilayers, each oscillation in the force profile indicates the force required to displace a single layer of water molecules from between the probe and bilayer. In contrast, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine fluid (Lalpha) phase bilayers at 60 degrees C and 1,2-dioleoyl-sn-glycero-3-phosphocholine fluid (Lalpha) phase bilayers at 24 degrees C seriously disrupt the molecular ordering of the water and result predominantly in a monotonic force profile.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Relationships between ice crystal size, water content and proton NMR relaxation times in cells

Biological specimens were frozen under controlled conditions. We questioned how the size of ice crystals, as measured in cryosectioned and cryoadsorbed sections of these biological specimens, relates to the water content and to the proton NMR relaxation times (T1 and T2) of the unfrozen specimens. The results permit the following conclusions: After rapid freezing in liquid propane cooled in a liquid nitrogen bath, the average size of ice crystals at distances of 150 microns or more from the surface of a particular tissue was always the same. Thus, the average size of the ice crystals was found to be characteristic of the type of biological tissue studied. Linear regression analysis showed average ice crystal size to have a significant correlation coefficient to T1 relaxation time and to water content. Specifically ice crystal size increased with T1 relaxation time and with water content. Multiple regression and path analysis demonstrated a positive correlation between the T1 relaxation time and the ice crystal size variation. Path analysis showed that both water content and T2 relaxation time were less directly correlated with ice crystal size. The findings from the path analysis and other observations show that the average size of ice crystals in subcellular compartments is best predicted by the proton T1 relaxation time. A working model is put forth to explain differences in ice crystal size observed between specimens enriched in globular or in parallel filamentous proteins.     

The water proton spin-lattice relaxation times in virus-infected cells.

The water proton spin-lattice relaxation times in HEp-2 cell cultures were determined immediately after 1 h of polio-virus adsorption. The shortening of the water T1 was closely related to the multiplicity of infection, allowing direct inspections of the virus--cell interaction since the first steps of the infectious cycle. Virus-induced structural and conformational changes of cell constituents were suggested to be detectable by NMR investigation of cell water.     

Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein/water system II - 13C NMR relaxation study

The addition of hydrophilic and hydrophobic molecules to the 1-monooleoyl glycerol (MO)/water (W) system has been investigated at a molecular level by 13C nuclear magnetic resonance (NMR) relaxation. Depending on the nature of the additive, the liquid crystalline phases of the MO/W binary system are modified. The 13C NMR spin lattice relaxation rates of the various MO carbons were determined in the presence of the additives for different types of L(2) and liquid crystalline phases. Data revealed that local dynamics are independent of type and amount of additive (within 5 wt.%), and also of the type of the structural arrangement. The curvature of the interface does not affect the local mobility of MO carbons, with the exception of the glycerol G3 and the carboxylic C1 carbons. Moreover, the presence of the double bond in the mid part of the hydrocarbon chain induces a levelling in the relaxation rates on the neighboring carbons. The 13C NMR spin lattice relaxation rates at two magnetic field strengths and the Overhauser enhancement were measured in the L(2) phase of the MO/W/sodium decanoate system. The use of a two-step model of relaxation allowed to estimate order parameters, and slow and fast motions of MO in the structured aggregate.     

Frequency dependence of water proton longitudinal NMR relaxation times in mouse tissues around the freezing transition

Water proton longitudinal NMR relaxation times were measured in various tissues of healthy and tumor-bearing mice. Measurements were performed as a function of the Larmor frequency nu in the range 6-90 MHz, and at two temperatures (theta + and theta -) bracketing the 'freezing transition', at which the major part of the water signal disappears. At both temperatures, 1/T1 behaves according to: 1/T1 = A/square root nu + B A and B are obtained at theta + and theta -, and yield the proportion of bound water, which is convincingly identified with non-freezable water. The proportions found lie around 6% for tumors and 12% for other tissues. Discrimination between tissues via T1 is demonstrated to be essentially due to the bound water proportion. Bound water on the one hand and free water on the other hand behave similarly in all tissues including tumors. The activation energy for free water is found to be identical to that of pure water, although relaxation times are markedly different. It is noticed that determining the bound water proportion by signal intensity measurements at theta + and theta - is less reliable than by the T1 method.     

A study of water autodiffusion in model biological membranes, oriented lipid bilayers

The autodiffusion of water in a multibilayer structure formed by dipalmitoyl phosphatidylcholine and oriented on glass plates was studied by the method of NMR with magnetic field pulse gradient. It was shown that water molecules occur in several states differing in the degree of interaction with lipid molecules. A spectrum of the coefficients of water autodiffusion in a direction transversal to bilayers was found. The use of samples with different distances between the plates and an analysis of the dependence of the mode of diffuse decay of spin echo on diffusion time and the orientation of the sample, as well as measurements at temperatures above and below the gel-liquid crystal phase transition in cholesterol-containing samples enabled one to discriminate the diffuse decay component responsible for the transbilayer movement of water. The coefficient of bilayer permeability was estimated using the Tanner model. It was shown that the formation of mechanical defects ("cracks") in plane oriented bilayers is the most probable reason for the presence of the water component with the relatively high coefficient of diffusion.     

Effect of dichloromethane on sickle cells. An in vitro water proton magnetic resonance relaxation study

To examine the manner in which dichloromethane inhibits sickling, sickle blood was subjected to both prevention and reversal schemes over a range of CH₂Cl₂ vapor pressures. Following CH₂Cl₂-treatment, the rotating frame spin lattice relaxation time (T_1?) of water protons in deoxygenated packed sickle cells was measured, cell types in a deoxygenated fixed sample were counted, and the extent of hemolysis determined. At CH₂Cl₂ vapor pressures above 200 mm, the NMR relaxation rate decreased sharply, the extent of hemolysis increased, the fraction of sickled cells and other abnormal erythrocytes decreased, and the fraction of biconcave discs increased. Apparently CH₂Cl₂ is absorbed by the cell membrane and preferentially lyses sickled cells and other abnormal cells. Part of the decrease in NMR relaxation rate with increased CH₂Cl₂ pressure is due to a larger fraction of discs, but an additional factor probably arises from CH₂Cl₂ inhibition of hemoglobin S gelation.