An NMR study of the temperature dependence of the coefficient of water self-diffusion through lipid bilayer membranes

The temperature dependence of the coefficient of water self-diffusion through plane-parallel lipid multilayers of the phospholipid dioleoylphosphatidylcholine oriented on a glass support has been studied in the temperature range of 20-60 degrees C by the method of NMR with magnetic field pulse gradient. The values of the coefficients of transbilayer water diffusion are by four orders of magnitude less than for bulky water and ten times less than the coefficients of lateral diffusion of the lipid under the same conditions. The temperature dependence of the coefficient of water diffusion is described by the Arrhenius law with an apparent activation energy of about 41 kJ/mol, which far exceeds the activation energy for the diffusion of bulky water (18 kJ/mol). The experimental data were analyzed using a "dissolving-diffusion" model, by simulating the passage of water through membrane channels, and by analyzing the exchange of water molecules in states with different modes of translation mobility, including pore channels and bilayer "defects". Each of the approaches used made it possible to take the significance of bilayer permeability for the apparent energy of activation of water diffusion into account and estimate the energies of activation of water diffusion in the hydrophobic moiety of the bilayer, which were found to be close to the values for bulky water. The coefficients of water diffusion in the system under examination and the coefficients of permeation of water through the bilayer were estimated, and the effect of bilayer "defects" on the coefficients of water diffusion along and across bilayers was studied.     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

A method for measuring sedimentation and diffusion of macromolecules in capillary tubes by total intensity and quasi-elastic light-scattering techniques.

Light scattered from a macromolecular solution in a capillary tube is used to determine both the sedimentation and translational diffusion coefficients. The capillary tube is spun in a preparative centrifuge, removed, and placed in a light-scattering photometer equipped with a scanning mechanism. The intensity distribution of scattered light along the tube represents the concentration profile in the tube and provides the measure of boundary migration. The sedimentation coefficient is determined from this measure and the applied centrifugal field. The diffusion coefficient is obtained from a time-autocorrelation analysis of fluctuations in intensity of light scattered from any fixed point of the profile. These coefficients were obtained for two monodisperse systems, R17 bacteriophage and 28s ribosomal rat liver RNA. The molecular weights obtained from ratios of these coefficients are in good agreement with literature values. In the sedimentation analysis, deviations from linearity between boundary displacement and applied field were found to be less than 1%. This precision confirms that the boundary is stable for the capillary geometry even in the absence of a preformed density gradient. The sedimentation coefficients of identical samples were also measured with the Spinco Model E analytical ultracentrifuge; results of the two methods agree to within 4%. As a consequence of the capillary tube geometry and light-scattering detection, sedimentation coefficients can be obtained from sample volumes of less than 100 μl. This detection techniques is thus far demonstrated to be at least an order of magnitude more sensitive than Schlieren optics, thereby useful when uv absorption is not applicable. For diffusion measurements there are also several inherent advantages. The diffusion coefficient is obtained from the identical sample, and scanning provides the capability to measure D from various parts of the sedimentation profiles and thereby directly explore concentration dependence, homogeneity, and integrity of the sample. The capillary tube with a layer of silicone oil over the sample and centrifugation provides an effective method to cleanse the solution and trap all dust.     

Study of catalytic site structure and diffusion of radicals in porous heterogenous systems with ESR,ENDOR and ESE

The generation of paramagnetic products by adsorption of quinones on activated catalysts has been used for the diagnostic of Lewis acid sites. It has been shown that the application of ENDOR, ESE, and 2 mm-band ESR are extremely effective methods for studying the nature of observed radical species and their environment. Two applications of the ESE method for studying the diffusion of spin probes in porous media are considered. The measurement of effective diffusion coefficients of radical probes in specimens of various heterogeneous systems is described. It has been found that effective diffusion coefficients depend strongly on the mean value of silica gel pore sizes and the mobility of the probe inside the pore.     

PFG-NMR measurements of the self-diffusion coefficients of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The self-diffusion coefficients for water in a series of copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA, swollen with water to their equilibrium states have been studied at 310 K using PFG-NMR. The self-diffusion coefficients calculated from the Stejskal-Tanner equation, D(obs), for all of the hydrated polymers were found to be dependent on the NMR storage time, as a result of spin exchange between the proton reservoirs of the water and the polymers, reaching an equilibrium plateau value at long storage times. The true values of the diffusion coefficients were calculated from the values of D(obs) in the plateau regions by applying a correction for the fraction of water protons present, obtained from the equilibrium water contents of the gels. The true self-diffusion coefficient for water in polyHEMA obtained at 310 K by this method was 5.5 x 10(-10) m(2)s-1. For the copolymers containing 20% HEMA or more a single value of the self-diffusion coefficient was found, which was somewhat larger than the corresponding values obtained for the macroscopic diffusion coefficient from sorption measurements. For polyTHFMA and copolymers containing less than 20% HEMA, the PFG-NMR stimulated echo attenuation decay curves and the log-attenuation plots were characteristic of the presence of two diffusing water species. The self-diffusion coefficients of water in the equilibrium-hydrated copolymers were found to be dependent on the copolymer composition, decreasing with increasing THFMA content.     

Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Determination of diffusion coefficients in a solution by the transport of a solute through porous membranes

The possibility of determining the coefficients of diffusion in solution by the transport of solutes through porous polymeric membrane was studied. Reliable and reproducible results can be obtained by using nucleoporous filters with cylindrical pores. The method enables the selective determining of the diffusion coefficients of solutes being in complex mixtures, which is of special interest for biochemical research. The possibilities of the method are illustrated on the pattern of some globular proteins, polyethylene glycols and proteolytic enzymes.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

Measuring the translational diffusion coefficients of small DNA molecules by capillary electrophoresis

The apparent translational diffusion coefficients of four 20 base pair (bp) DNA oligonucleotides with different sequences have been measured by capillary electrophoresis, using the stopped migration method. The diffusion coefficients of the four oligomers were equal within experimental error, and averaged (120 +/- 10) x 10(-8) cm(2) s(-1) in 40 mM Tris-acetate-EDTA buffer at 25 degrees C. Since this value is nearly identical to the translational diffusion coefficient determined for a different 20-bp oligomer using other methods, the stopped migration method can accurately measure the diffusion coefficients of small DNA oligomers. The apparent diffusion coefficient of a 118-bp DNA restriction fragment was also measured by the stopped migration method. However, the observed value was approximately 25% larger than expected from other measurements, possibly because the diffusion coefficients of larger DNA molecules are somewhat dependent on the ionic strength of the solution.     

The distribution of serum albumin in human normal and degenerate articular cartilage

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymatic methods. With naturally occuring cartilage large a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partitiion coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

The distribution of serum albumin in human normal and degenerate articular cartilage.

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymic methods. With naturally occurring cartilage a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partition coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Diffucison of OXYGEN, Nitrogen and water in hyluronate solutions.

A method is reported for the measurement of the diffusion coefficients in water of some sparingly soluble gases. The results obtained for the diffusion coefficients of oxygen and nitrogen gas through water at 25 degrees C are 2.12. 10-5 and 2.61 . 10-5 cm-2 . s-1, respectively. A check on the accuracy of the teachnique using tritiated water as the diffusing substance gave a value of 2.15 . 10-5 cm-2 . s-1 which agrees within 3% with recent values from the literature. The method was applied to the measurement of oxygen, nitrogen, and tritiated water diffusion coefficients through agarose gels and through agarose gels containing hyluronate. The results indicate that the hyaluronate had only a small effect as a barrier to the diffusion of such low molecular weight substance.     

Conformational studies of BSA using laser light scattering

The usefulness of laser light scattering as a technique for determining protein conformation has been investigated by studying the self-association and drug binding of bovine serum albumin (BSA). The diffusion coefficients of BSA monomers and dimers have been measured and the ratio of these two quantities indicates that in the dimer, the subunit separation is 2.2 times the monomeric hydrodynamic radius. The binding of salicylate to BSA causes an increase in its diffusion coefficient corresponding to a reduction in the frictional drag of the solvent on the protein molecules. It has been found that data obtained using laser light scattering may be interpreted confidently only when proper care has been taken in sample preparation and the scattered intensity autocorrelation function has been appropriately analyzed.     

Sizes and mass distributions of clathrin-coated vesicles from bovine brain

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

A simple method for estimating cytoplasmic diffusion coefficients

The diffusion coefficients of radioactively labelled substances in cytoplasm or other fluids are determined in vitro. The fluid containing the labelled substance is filled into a cylinder with one open end, through which the labelled substance diffuses out into a stirred outer medium. The diffusion coefficient is calculated by a one-dimensional diffusion equation from the rate of loss from the cylinder, and the length of the cylinder. The diffusion coefficients of tritiated water in several fluids have been determined. The results are in good agreement with those obtained by other methods.     

Determining diffusion coefficients in inhomogeneous tissues using fluorescence recovery after photobleaching

Diffusion plays an important role in the transport of nutrients and signaling molecules in cartilaginous tissues. Diffusion coefficients can be measured by fluorescence recovery after photobleaching (FRAP). Available methods to analyze FRAP data, however, assume homogeneity in the environment of the bleached area and neglect geometrical restrictions to diffusion. Hence, diffusion coefficients in inhomogeneous materials, such as most biological tissues, cannot be assessed accurately. In this study, a new method for analyzing data from FRAP measurements has been developed, which is applicable to inhomogeneous tissues. It is based on a fitting procedure of the intensity recovery after photobleaching with a two-dimensional finite element analysis, which includes Fick's law for diffusion. The finite element analysis can account for distinctive diffusivity in predefined zones, which allows determining diffusion coefficients in inhomogeneous samples. The method is validated theoretically and experimentally in both homogeneous and inhomogeneous tissues and subsequently applied to the proliferation zone of the growth plate. Finally, the importance of accounting for inhomogeneities, for appropriate assessment of diffusivity in inhomogeneous tissues, is illustrated.     

Molecular simulation study of structure and dynamical properties of nitrate anion in sodium chloride aqueous solution

The molecular dynamics simulations have been performed for the sodium nitrate solution at various concentrations in the 1.1 mol% sodium chloride aqueous solution, which is a model of the sea water. The structure, the velocity correlation functions and the frequency-dependent diffusion coefficients have been obtained. The share viscosity has also been obtained at various concentrations using the Green–Kubo formula, which agrees well with the experimental data. The complex formation and the microscopic charge neutrality in the solution have been discussed in relation to the ionic conductivity estimated by the Nernst–Einstein relation.     

A quasi-steady-state approximation method for diffusion problems: I. Concentration dependent diffusion coefficients

A generalization of Landahl's approximation method (H. D. Landahl,Bull. Math. Biophysics,15, 49–61, 1953) for non-linear diffusion problems is suggested. The method is applied to sorption, desorption, and free diffusion problems involving concentration-dependent diffusion coefficients. With some limitations, the results compare favorably with those obtained by numerical methods.     

Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media.

A new method for the measurement of diffusion in thick samples is introduced, based upon the spatial Fourier analysis of Tsay and Jacobson (Biophys. J. 60: 360-368, 1991) for the video image analysis of fluorescence recovery after photobleaching (FRAP). In this approach, the diffusion coefficient is calculated from the decay of Fourier transform coefficients in successive fluorescence images. Previously, the application of FRAP in thick samples has been confounded by the optical effects of out-of-focus light and scattering and absorption by the sample. The theory of image formation is invoked to show that the decay rate is the same for both the observed fluorescence intensity and the true concentration distribution in the tissue. The method was tested in a series of macromolecular diffusion measurements in aqueous solution, in agarose gel, and in simulated tissue consisting of tumor cells (45% v/v) and blood cells (5% v/v) in an agarose gel. For a range of fluorescently labeled proteins (MW = 14 to 600 kD) and dextrans (MW = 4.4 to 147.8 kD), the diffusion coefficients in aqueous solution were comparable to previously published values. A comparison of the spatial Fourier analysis with a conventional direct photometric method revealed that even for the weakly scattering agarose sample, the conventional method gives a result that is inaccurate and dependent on sample thickness whereas the diffusion coefficient calculated by the spatial Fourier method agreed with published values and was independent of sample thickness. The diffusion coefficient of albumin in the simulated tissue samples, as determined by the spatial Fourier analysis, varied slightly with sample thickness.(ABSTRACT TRUNCATED AT 250 WORDS)