Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.     

Xylem pressure response in maize roots subjected to osmotic stress: determination of radial reflection coefficients by use of the xylem pressure probe

The absolute pressure in conducting xylem vessels of roots of 2-week-old, slowly transpiring intact maize plants (bathed in nutrition medium) was determined to be +0·024 ± 0·044 MPa using the xylem pressure probe. When the roots were subjected to osmotic stress (NaCI, KCI or sucrose), the xylem pressure decreased immediately and became more negative. However, the response of xylem pressure to osmotic stress was considerably attenuated, indicating that the radial reflection coefficients, σ₁₃ of the maize root for these solutes were rather low (between 0·2 and 0·4 depending on the concentration of the osmoticum). The low values of a, may be caused (partly) by unstirred layer effects. In repeated osmoticum/nutrition regimes a complex pattern of changes in xylem pressure was observed which was apparently linked to the interplay between transpiration and (passive and/or active) solute loading of the xylem. These processes were not observed when the roots were subjected to osmotic stress after excision. In this case, a biphasic response was observed comparable to that found for excised roots using the root pressure probe.     

Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography

The importance of weak protein interactions, such as protein self-association, is widely recognized in a variety of biological and technological processes. Although protein self-association has been studied extensively, much less attention has been devoted to weak protein cross-association, mainly due to the difficulties in measuring weak interactions between different proteins in solution. Here a framework is presented for quantifying the osmotic second virial cross coefficient directly using a modified form of self-interaction chromatography called cross-interaction chromatography. A theoretical relationship is developed between the virial cross coefficient and the chromatographic retention using statistical mechanics. Measurements of bovine serum albumin (BSA)/lysozyme cross-association using cross-interaction chromatography agree well with the few osmometry measurements available in the literature. Lysozyme/alpha-chymotrypsinogen interactions were also measured over a wide range of solution conditions, and some counterintuitive trends were observed that may provide new insight into the molecular origins of weak protein interactions. The virial cross coefficients presented in this work may also provide insight into separation processes that are influenced by protein cross-interactions, such as crystallization, precipitation, and ultrafiltration.     

Determination of reflection coefficients for various ions and neutral molecules in sarcoplasmic reticulum vesicles through osmotic volume change studied by stopped flow technique

Osmotic volume change of sarcoplasmic reticulum vesicles was studied by following the change in light-scattering intensity using a stopped flow apparatus. From the analysis of the initial rate of scattering change, reflection coefficients for various ions and neutral molecules were determined. The following are typical results: K+, 0.72; Tris+, 0.98; choline 1; NO3-, 0.32; Cl-, 0.46; methanesulfonate, 0.62; gluconate, 0.96; glycerol, 0.86; and glucose, 1. When the K+ permeability was increased in the presence of 10(-6) g valinomycin/ml, the reflection coefficient for K+ changed from 0.72 to 0.31. It was found that there was a close relationship between the reflection coefficients and the permeabilities of the solutes. Hydraulic conductivity was also determined from the initial rate of light scattering change and was not different for the different solutes. The water permeability was estimated to be 2.1 x 10(-3) cm/sec at 23 degrees C.     

The osmotic coefficients of the sodium form of some biopolymers

The osmotic coefficients phi p,Na of dilute solutions of the sodium form of some weakly acidic polymers are theoretically predicted in this work. Based on the measured value 0.73 of gamma Na, the activity coefficient of free Na+, of the completely ionized humic acid (sodium salt) in a salt-free solution, the effective interligand distance b is calculated to be 11.34 A by using Manning's counterion condensation theory [Manning, G. S. (1969) J. Chem. Phys. 51(3), 924]. The corresponding values of gamma Na (measured experimentally) and b for the completely ionized exopolymer of Pseudomonas atlantica are 0.624 and 7.57 A when cultivated at a dilution rate D = 0.015 h-1, 0.647 and 8.19 A at D = 0.025 h-1, and 0.613 and 7.29 Aat D = 0.06 h-1. For alginic acid (in the completely ionized sodium form), gamma Na = 0.40 and b = 4.71 A. The osmotic coefficients phi p,Na for the partially and the completely ionized polymers are then predicted with Manning's theory as well.     

Direct method for osmotic pressure measurement (author's transl)]

A new dynamic method to measure osmotic pressure was presented. The method is easy and with only a 1.8% degree of error. All determinations were done with an osmometer, equipped with three small chambers of methyl metacrilate and dialyser membranes. The central area had two capillaries: one for measuring and the other for changing dynamic pressure. The data from different albumin concentrations were satisfactory, and they agreed with published results from others. The present results indicate that osmotic pressure is an exponential function of albumin concentration.     

Determination of the hydraulic conductivity and of reflection coefficients in Nitella flexilis by means of direct cell-turgor pressure measurements

From direct and continuous measurements of the internal hydrostatic pressure (P) in the internodes of Nitella flexilis, the reflection coefficients (σ_s) of some non-electrolytes were determined, using a zero-flow method, and were compared with those found previously on Valonia utricularis and with those obtained by Dainty and Ginzburg on other Characean internodes from transcellular osmosis experiments. The hydraulic conductivities (Lp) of the cell membranes were determined by two independent methods, that is, using hydrostatically or osmotically induced flows. From the exponential time course of P in such experiments and from the volumetric elastic modulus (ε) of the cell wall, Lp was calculated. The effect of unstirred layers in the methods described was negligibly small.In osmotic experiments with different non-plasmolysing external sucrose concentrations (20–200 mM) the exosmotic hydraulic conductivity (Lp_ex) decreases markedly with increasing concentration, while the endosmotic hydraulic conductivity (Lp_en) shows only a weak dependence. In the hydrostatic experiments the hydraulic conductivities for single cells were constant in the pressure range for P from 2 to 7 atm. In this pressure range Lp_en and Lp_ex varied for different cells from 2.2·10^?5 to 2.8·10^?5 and from 1.8·10^?5 to 2.5·10^?5 cm·s^?1·atm^?1, respectively, with an average ratio Lp_en to Lp_ex of 1.1, which indicates a polarity in water movement.These values were the same as those obtained in the osmotic experiments from extrapolation to zero sucrose concentration. At internal pressures below 2 atm the Lp-values markedly increase on approaching the plasmolytic point.The results are discussed in terms of a dehydration of the membranes (or the cytoplasm) at increased solute concentrations. In addition, the strong dependence of Lp at low internal hydrostatic pressures points to a direct influence of P on the water permeability of the membranes.     

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Colloid osmotic pressure: its measurement and clinical value.

Plasma colloid osmotic pressure (COP) is an important determinant in the appearance of edema. The development of a simple technique for COP measurement, based on an electronic pressure transducer and a semipermeable membrane system, has led to an appreciation of the value of COP determinations in clinical practice. In a steady state the measured COP replicates the value computed from serum proteins. In pathologic sera a derived value is unreliable. The normal human plasma COP averages 25.4 mm Hg. This value tends to decrease with age, is lower in females and is also lower in subjects at bed rest. As a clinical tool COP measurement represents an unduplicated contribution to the differential diagnosis of pulmonary edema. In critically ill patients COP measurement represents a reliable predictor of survival.     

Measurement of osmotic second virial coefficients by zonal size-exclusion chromatography

Numerical simulation of protein migration reflecting linear concentration dependence of the partition isotherm has been used to invalidate a published procedure for measuring osmotic second virial coefficients (B₂₂) by zonal exclusion chromatography. Failure of the zonal procedure to emulate its frontal chromatographic counterpart reflects ambiguity about the solute concentration that should be used to replace the applied concentration in the rigorous quantitative expression for frontal migration; the recommended use of the peak concentration in the eluted zone is incorrect on theoretical grounds. Furthermore, the claim for its validation on empirical grounds has been traced to the use of inappropriate B₂₂ magnitudes as the standards against which the experimentally derived values were being tested.     

Continuous measurement of protein osmotic pressure in blood and lymph of sheep

We have continuously measured protein osmotic pressure of blood and lymph in sheep to compare two kinds of needle osmometers (rigid and flexible) with a membrane osmometer (Wescor). We also compared the averaged values of the continuous measurement with osmotic pressure calculated from total protein and albumin fraction, using the Yamada equation. The rigid-needle and membrane osmometers showed excellent correlation (y = 1.00x + 0.06; r greater than 0.99). The flexible-needle osmometer tended to overestimate osmotic pressure (avg 16%). We used the rigid-needle osmometer for continuous measurements of protein osmotic pressure of blood and lymph in anesthetized or unanesthetized sheep to observe changes in protein osmotic pressure of blood and lymph through the three different interventions. The relationship between the theoretical values (x) and the continuous measurements (y) of osmotic pressure was good (y = 0.99x + 0.16, r = 0.97), but after various interventions, the continuously measured protein osmotic pressure tended to exceed the calculated measurements. The continuous measurement should be monitored with spot samples measured in a stationary osmometer or by calculation of osmotic pressure from total protein concentration and albumin fraction.