In vitro effects of ozone on human erythrocyte membranes: An EPR study

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20°C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40°C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37°C and 16°C were observed

Evidence of Surface Diffusion of Water Molecules on Proteins of Rabbit Lens by ¹H NMR Relaxation Measurements

In this work, we propose a relaxation model for the interpretation of NMR proton spinlattice and spin-spin relaxation times of mammalian lenses. The framework for this model is based on nuclear magnetic spin-lattice relaxation measurements as a function of temperature at different Larmor frequencies for whole rabbit lenses and fragments of the lens. According to this model, two different dynamic processes of the water molecules determine the relaxation behaviour, namely rotational diffusion and translational surface diffusion. These dynamic processes in conjuction with a two site exchange model give a good explanation of all the measured relaxation data. From the experimental data, we were able to obtain the activation parameters for rotational and translational diffusion of bound lens water. Correlation times of 2.1x10⁻¹¹ sec and 2.5x10⁻⁹ sec and activation energies of 20.5 kJ/mol and 22.5 kJ/mol respectively were found at 308K. At low Larmor frequencies (≤100 MHz) the longitudinal relaxation is mainly determined by translational surface diffusion of bound water with a mean square displacement of 1.5 nm, whereas at higher frequencies (≥300 MHz), rotational diffusion is the main relaxation mechanism. The spin-spin relaxation is determined by translational diffusion over the whole frequency range and therefore shows only a very small dispersion. By our model it is possible to explain: 1) the strikingly large difference between the T₁ value and the T₂A and T₂B values observed in the lens and 2) the different values of the activation energies measured at different fields for the lens

Evidence of Anisotropie Reorientations of Water Molecules in the Cortex of the Rabbit Lens Detected by ¹H-NMR Spectroscopy

We studied the order of water molecules in different fragments of nucleus and cortex of lenses of 3 month old rabbits by investigating the ¹H-NMR spin-spin relaxtion behaviour of the water protons at room temperature. The experiments were carried out using the Carr Purcell Meiboom Gill (CPMG) technique. The apparent relaxation rate was found to be dependent on the pulse spacing in the CPMG sequence in a different way for nucleus and cortex. While for the nucleus the pulse spacing dependence can be explained by chemical exchange of water protons, the pulse spacing dependence of the cortex protons suggests the existence of not fully averaged residual magnetic dipolar couplings among the protons of a water molecule. To support this interpretation, measurements of the ratio of the solid echo (90x—90y) to the Hahn echo (90x—180x) amplitude were carried out for the same samples. These experiments give the expected ratio of = 0.5, characteristic of no residual couplings for the nucleus, but a ratio of = 0.7 for the cortex, which is characteristic of residual dipolar couplings, caused by anisotropie reorientations of the water molecules. Thus, evidence for an ordered state of the water molecules in the cortex has been found

¹H NMR and Calorimetric Measurements on Rabbit Eye Lenses

The dynamic properties of water molecules in the rabbit lens were studied by proton nuclear magnetic resonance line shape analysis, measurements of relaxation times as a function of temperature and calorimetric measurements. The experiments prove, as already suggested by other authors, that there are two types of water in the lens of rabbit eyes, namely bound unfreezable hydration water and bulk freezable water. Line shape analysis and relaxometry showed, that this two types of water exist in two different environments, which may be identified as the nucleus and the cortex of the lens. The line shape analysis showed furthermore that water molecules in the rabbit lens has a common spin lattice relaxation time (T₁), but two different transverse relaxation times (T₂A and T₂B). The tentative model of fast water exchange on the T₁ time scale and slow water exchange on the T₂ time scale, was used to explain experimental proton relaxation data of the rabbit lens. An estimation for this exchange rate kₑₓ by comparing it to the relaxation times is given (T₁⁻¹≪kₑₓ≪ T₁⁻¹). It has also been shown by a calorimetric measurements, that the lenses can be easily undercooled to temperatures well below the freezing point of water. The achievable maximum undercooling temperature of the lens is a function of the cooling rate KC, therefore it has to be considered as an experimentally adjustable parameter which is not characteristic for the investigated sample. Thus it must be noted that any previous discussions about the specific value of the temperature of water crystallisation in biological systems need to be carefully reconsidered