The osmotic pressure of chondroitin sulphate solutions: experimental measurements and theoretical analysis

We used equilibrium dialysis to measure the osmotic pressure of chondroitin sulphate (CS) solutions as a function of their concentration and fixed charge density (FCD) and the ionic strength and composition of the solution. Osmotic pressure varied nonlinearly with the concentration of chondroitin sulphate and in 0.15 M NaCl at FCDs typical of uncompressed cartilage (approximately 0.4 mmol/g extrafibrillar H2O) was approximately 3 atmospheres. Osmotic pressure fell by 60% as solution ionic strength increased up to about 1 M, but remained relatively constant at higher ionic strengths. The ratio of Ca2+ to Na+ in the medium was a minor determinant of osmotic pressure. The data are compared with a theoretical model of the electrostatic contribution to osmotic pressure calculated from the Poisson-Boltzmann equation using a rod-in-cell model for CS. The effective radius of the polyelectrolyte rod is taken as a free parameter. The model qualitatively reproduces the non-linear concentration dependence, but underestimates the osmotic pressure by an amount that is independent of ionic strength. This difference, presumably arising from oncotic and entropic effects, is approximately 1/3 of the total osmotic pressure at physiological polymer concentrations and ionic strength.     

Plasma protein osmotic pressure equations and nomogram for sheep

The equations developed by Landis and Pappenheimer (Handbook of Physiology. Circulation, 1963, p. 961-1034) for calculating the protein osmotic pressure of human plasma proteins have been frequently used for other animal species without regard to the fractional albumin concentration or correction for protein-protein interaction. Using an electronic osmometer, we remeasured the protein osmotic pressure of purified sheep albumin and sheep plasma partially depleted of albumin. We measured protein osmotic pressures of serial dilutions over the concentration range 0-180 g/l for albumin and 0-100 g/l for the albumin-depleted proteins at room temperature (26 degrees C). Using a nonlinear least squares parameter-fitting computer program, we obtained the equation of best fit for purified albumin, and then we used that equation together with the measured albumin fraction to obtain the best-fit equation for the nonalbumin proteins. The equation for albumin is IIcmH2O,39 degrees C = 0.382C + 0.0028C2 + 0.000013C3, where C is albumin concentration in g/l. The equation for the nonalbumin fraction is IIcmH2O,39 degrees C = 0.119C + 0.0016C2. Up to 200- and 100-g/l protein concentration, respectively, these equations give the least standard error of the estimate for each of the virial coefficients. The computed number-average molecular weight for the nonalbumin proteins is 222,000. Using the new equations, we constructed a nomogram, based on the one of Nitta and co-workers (Tohoku J. Exp. Med. 135: 43-49, 1981). We tested the nomogram using 144 random samples of sheep plasma and lymph from 31 sheep. We obtained a correlation coefficient of 0.99 between the measured and nomogram estimates of protein osmotic pressure.     

Hypotension with intravenous immunoglobulin therapy: importance of pH and dimer formation

Therapy with intravenous immunoglobulin preparations has been used effectively in a wide range of conditions. Although generally well tolerated, intravenous immunoglobulin preparations may be associated with transient hypotension in some patients. This study examined the role of different immunoglobulin G fractions in the development of intravenous immunoglobulin-induced hypotension in an anaesthetized rat model and assessed the effects of a new liquid immunoglobulin prepared at a low pH on both the formation of immunoglobulin G dimers and the development of hypotension. The effects of this new preparation in an experimental autoimmune encephalomyelitis model were also evaluated.Results from the haemodynamic studies indicated that immunoglobulin G dimers in polyclonal immunoglobulin G are responsible for the hypotensive events associated with some immunoglobulin preparations. They also showed that adjustment to an acidic pH results in the rapid dissociation of immunoglobulin G dimers and prevents the development of hypotension. Additional experiments demonstrated that only immunoglobulin G dimers with a functional Fc fragment can bind to Fcgamma receptors on macrophages to induce the release of blood pressure-lowering mediators. Moreover, essentially monomeric Fc fragments can block the blood pressure-lowering effects of immunoglobulin G dimers.Preparation of a new liquid intravenous immunoglobulin with the pH adjusted to 4.3 prevents the formation of immunoglobulin G dimers even over long-term storage and does not significantly affect blood pressure in a rat model. This preparation is as effective as other intravenous immunoglobulin preparations in ameliorating symptoms of experimental autoimmune encephalomyelitis. These results, like those from previous studies, indicate that preparation of intravenous immunoglobulin at a low pH substantially reduces immunoglobulin G dimerization; this effect significantly decreases the potential for intravenous immunoglobulin to induce hypotension without reducing its clinically relevant biological activity.     

Regulation of glycosaminoglycan function by osmotic potentials. Measurement of water transfer during antithrombin activation by heparin.

The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.     

The glass transition behavior of the globular protein bovine serum albumin

The glass-like transition behavior of concentrated aqueous solutions of bovine serum albumin was examined using rheological techniques. At mass fractions >0.4, there was a marked concentration dependence of viscosity with a glass-like kinetic arrest observed at mass fractions in the region of 0.55. At mass fractions >0.6 the material behaved as a solid with a Young's modulus rising from approximately 20 MPa at a mass fraction of 0.62-1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 20 degrees C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres.     

响应面法优化酶法转化合成维生素C糖苷（AA-2G）条件的研究

采用Design—Expert软件的Central Composite Design（CCD）响应面设计对环糊精葡萄糖苷转移酶转化合成糖基抗坏血酸（AA-2G）的五个主要因素（转化时间、转化温度、pH、Vc浓度、β-环糊精浓度）进行了研究。采用降维分析方法对pH与转化时间、转化温度、Vc浓度、β-环糊精浓度以及反应温度与反应时间的交互作用对酶法转化合成AA-2G的影响进行了分析。建立了影响因素与响应值之间的回归方程,根据回归方程优化得到最佳转化条件为：转化时间25h,温度36．5℃,pH5．4,Vc72dL,β-环糊精55g／L。在此条件下,AA-2G的理论产量为10．06g／L,在验证实验中AA-2G的产量为9．76g／L,与预测的理论产量接近,比优化前提高了33％。     

Chromium Phytoaccumulation from Solution by Selected Hydrophytes

There is increasing interest in the role of wetland plants in the aquatic phytoremediation of toxic metals. In this experiment, we evaluate the Cr removal capacity of four hydrophyte species (Nasturtium officinale L., Veronica beccabunga L., Mentha longifolia L., R.Br., Cardamine uliginosa L.) under varying nutritional conditions (full-strength and half-strength solution cultures), and over a range of Cr concentration (0, 2, 4, 6, and 8 mg L^-1). The results indicate that Cr accumulation is affected by both initial Cr concentration and strength of the nutrient solution. Phytoaccumulation increased with initial Cr concentration and plants grown in the full-strength solution accumulated more Cr at the higher initial solution concentration. Cr was predominantly accumulated in the roots, with minimal shoot translocation, which limits the hazard of Cr entering the food chain through ingestion by animals. Accumulation was large and reached up to 6700 mg Cr Kg^-1 in the roots of Veronica beccabunga.     

The effect of ionic strength, temperature, and pressure on the interaction potential of dense protein solutions: from nonlinear pressure response to protein crystallization

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.     

Self-association of lysozyme. Thermochemical measurements: effect of chemical modification of Trp-62, Trp-108, and Glu-35.

Heats of dilution and of saccharide binding for hen egg white lysozyme have been measured at 30 degrees, 0.1 ionic strength, and pH 7 over the range 3 to 95 mg of protein/ml. The concentration dependence of the apparent relative molar enthalpy of lysozyme derived from these results gives the thermodynamic parameters for the formation of an intermolecular contact in an indefinite (head-to-tail) self-association process as delta G 0 = -3.9 kcal/mol, delta H 0 = -6.4 kcal/mol, and delta S 0 = -8,3 e.u. Oxindolealanine-62-lysozyme does not undergo self-association reactions that can be detected calorimetrically. This derivative reacts with native lysozyme to form hybrid polymeric species with free energy and enthalpy of interaction similar to those for the polymers of native lysozyme. These results are consistent with the intermolecular contact in the self-assocaition of lysozyme being asymmetric (head-to-tail). The heat of dilution of the derivative of lysozyme in which Glu-35 is blocked as the ester with oxindolealanine-108 is like that observed for native lysozyme in acid solution and is independent of pH. The concentration difference spectrum that develops through self-association is of the shape expected for introduction of an indole chromophore into a charge-free region of the intermolecular contact. The foregoing results indicate that Glu-35 and Trp-62 are part of the contact, that perturbation of Trp-108 does not make a principle contribution to the concentration difference spectrum, and that no acid group other than Glu-35 is perturbed by self-association. There is a small change in the heat of (GlcNAc)3 binding over the range 0.005 to 0.034 M saccharide. These data give the value of -1 kcal/mol for the enthalpy change for formation of the 2:1 saccharide-enzyme complex (ES2) from ES and S.     

Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata : Libellulidae)

Larvae of the widespread dragonfly, Libellula quadrimaculata, were adapted to a series of salt solutions, and the osmotic pressure, and sodium, potassium and chloride concentrations in the haemolymph measured. The regulation of potassium is extremely efficient over the range 0–50 m-mole/l. external concentration. Above this, larvae die. Sodium and chloride are regulated to a lesser extent, the larvae being able to withstand considerable changes in the concentration of these ions in the haemolymph. However, at higher external concentrations, the haemolymph concentration of these ions is maintained below that of the external medium. The osmotic pressure is regulated in parallel with sodium concentration over most of the range tested. However, in higher salinities, the osmotic pressure of the haemolymph does not fall below that of the external medium. This is seen as a strategy to limit the amount of drinking in saline media. Overall, the osmoregulatory system of L. quadrimaculata resembles that of brackish-water insects, rather than that of the more strictly freshwater dragonflies that have been studied.     

The role of mechanical pressure difference in the generation of membrane voltage under conditions of concentration polarization

The mechanical pressure difference across the bacterial cellulose membrane located in a horizontal plane causes asymmetry of voltage measured between electrodes immersed in KCl solutions symmetrically on both sides of the membrane. For all measurements, KCl solution with lower concentration was above the membrane. In configuration of the analyzed membrane system, the concentration boundary layers (CBLs) are created only by molecular diffusion. The voltages measured in the membrane system in concentration polarization conditions were compared with suitable voltages obtained from the model of diffusion through CBLs and ion transport through the membrane. An increase of difference of mechanical pressure across the membrane directed as a difference of osmotic pressure always causes a decrease of voltage between the electrodes in the membrane system. In turn, for mechanical pressure difference across the membrane directed in an opposite direction to the difference of osmotic pressure, a peak in the voltage as a function of mechanical pressure difference is observed. An increase of osmotic pressure difference across the membrane at the initial moment causes an increase of the maximal value of the observed peak and a shift of this peak position in the direction of higher values of the mechanical pressure differences across the membrane.     

Sedimentation equilibrium in a solution containing an arbitrary number of solute species at arbitrary concentrations: theory and application to concentrated solutions of ribonuclease

Simple expressions are derived describing the equilibrium concentration gradient of each species in a solution containing an arbitrary number of solute species at arbitrary concentration, as a function of the concentration of all species. Quantitative relationships between the species gradients and experimentally observable signal gradients are presented. The expressions are model-free and take into account both attractive and repulsive interactions between all species. In order to analyze data obtained from strongly nonideal solutions, a statistical thermodynamic model for repulsive solute-solute interactions is required. The relations obtained are utilized to analyze the dependence of the equilibrium gradient of ribonuclease A in phosphate-buffered saline, pH 7.4, upon total protein concentration. Experimental results are interpreted in the context of a model for weak self-association leading to the formation of significant amounts of oligomers at total protein concentrations exceeding 25 g/l.     

The Donnan Equilibrium: A Theoretical Study of the Effects of Interionic Forces

We investigate the conditions under which nonideality in solution influences the Donnan equilibrium. Of the various parameters that characterize this equilibrium, the osmotic pressure established across the Donnan membrane is found to be particularly sensitive to intermolecular interactions between the diffusible and nondiffusible ionic species. Under physiologically appropriate conditions, we find that it is almost never valid to use Debye-Hückel theory to calculate ionic activities: it is important to take proper account of ion size. When the diffusible species is a 1-1 electrolyte, this can be done using the mean spherical approximation (MSA) for a mixture of ions of different diameters. For 2-2, or higher-valent, electrolytes one should also include the effects of the second ionic virial coefficient, which the MSA omits.     

Static light scattering from concentrated protein solutions, I: General theory for protein mixtures and application to self-associating proteins

Exact expressions for the static light scattering of a solution containing up to three species of point-scattering solutes in highly nonideal solutions at arbitrary concentration are obtained from multicomponent scattering theory. Explicit expressions for thermodynamic interaction between solute molecules, required to evaluate the scattering relations, are obtained using an equivalent hard particle approximation similar to that employed earlier to interpret scattering of a single protein species at high concentration. The dependence of scattering intensity upon total protein concentration is calculated for mixtures of nonassociating proteins and for a single self-associating protein over a range of concentrations up to 200 g/l. An approximate semiempirical analysis of the concentration dependence of scattering intensity is proposed, according to which the contribution of thermodynamic interaction to scattering intensity is modeled as that of a single average hard spherical species. Simulated data containing pseudo-noise comparable in magnitude to actual experimental uncertainty are modeled using relations obtained from the proposed semiempirical analysis. It is shown that by using these relations one can extract from the data reasonably reliable information about underlying weak associations that are manifested only at very high total protein concentration.     

Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies.

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q--immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15--0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9--12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338--344].     

Phase behavior and hydration of silk fibroin

The osmotic stress method was applied to study the thermodynamics of supramolecular self-assembly phenomena in crystallizable segments of Bombyx mori silkworm silk fibroin. By controlling compositions and phases of silk fibroin solution, the method provided a means for the direct investigation of microscopic and thermodynamic details of these intermolecular interactions in aqueous media. It is apparent that as osmotic pressure increases, silk fibroin molecules are crowded together to form silk I structure and then with further increase in osmotic pressure become an antiparallel beta-sheet structure, silk II. A partial ternary phase diagram of water-silk fibroin-LiBr was constructed based on the results. The results provide quantitative evidence that the silk I structure must contain water of hydration. The enhanced control over structure and phase behavior using osmotic stress, as embodied in the phase diagram, could potentially be utilized to design a new route for water-based wet spinning of regenerated silk fibroin.     

中华蟾蜍蝌蚪对高渗溶液的耐受性

为了解中华蟾蜍蝌蚪对高渗溶液的耐受性,采用常规静态实验,用蔗糖调节溶液渗透压对蟾蜍蝌蚪进行渗透耐受实验;采用方差分析统计溶液渗透压对蟾蜍蝌蚪的存活率、体积变化和活动性的影响。结果显示,蟾蜍蝌蚪能够忍受溶液渗透压在一定范围内的变化,对渗透压较低的溶液其适应对策为调节,而对渗透压较高的溶液则为协变。蝌蚪对水溶液的耐受渗透压为1485．24mOsm,其存活率随渗透压增高而降低;同一渗透压下,蝌蚪存活率随在溶液中暴露时间的延长而降低。蟾蜍蝌蚪体内水分在不同浓度溶液中丧失的程度存在显著差异,即使在同一浓度的溶液中,体积会随时间的增加而不断缩小,并且溶液浓度越高,体积减小的速率越大。身体失水对蝌蚪的行为性会产生一定的影响,蝌蚪的活泼性随时间的延长而降低。     

High osmotic stress behavior of hyaluronate and heparin.

Using polyethylene glycol and dextran as osmotic stressing agents, the concentrations of hyaluronate and heparin were measured as a function of osmotic pressure II over the range of 0.03 to nearly 50 atmospheres. The experimental results were analyzed in terms of the Donnan osmotic pressure, the virial expansion, and Flory's first neighbor interaction parameter. In addition, II was looked at as a function of the reciprocal cube root of the concentration, which represents an average intermonomer spacing at high concentrations. The decay lengths in the so-called hydration region were found to be around 2.6 A and negligibly salt dependent. In the electrostatically dominated region the decay lengths were found to be dependent on the ionic strength, but not simply so. The osmotic compressibilities were also calculated, and were compared to compressibility data of corneal stroma and articular cartilage. These latter compressibilities were close to those for the pure hyaluronate and heparin, strengthening the evidence that glycosaminoglycans (GAGs) are largely responsible for connective tissue compressibility. Higher compressibilities for previously reported GAG data is thought to be related to the protein content of those samples.