Role of protein conformation and aggregation in pumping water in and out of a cell.

Dialysis cassettes containing BSA solutions were used to simulate passive in vivo conditions to assess the effect of protein conformation and aggregation on cell water content. The cassettes were suspended in dextran solutions to provide a range of fixed osmotic stress values simulating blood plasma. The system was placed on a shaker for 24 h to attain equilibrium. Four manipulation methods; pH, cosolute salt concentration, e.g. NaCl, temperature annealing and urea concentration denaturant were varied to produce well-known manipulations of BSA conformation. It was observed that the cell water content varied from +14% to about -13% with changes in protein conformation and aggregation. The findings demonstrate that a change in protein conformation and aggregation, pumps water in and out of a cell to maintain equilibrium % water content matching the protein conformational hydration parameter. This concept supplements existing theories on cell volume regulation.

A mechanistic view of the non-ideal osmotic and motional behavior of intracellular water.

It is commonly assumed that essentially all of the water in cells has the same ideal motional and colligative properties as does water in bulk liquid state. This assumption is used in studies of volume regulation, transmembrane movement of solutes and electrical potentials, solute and solution motion, solute solubility and other phenomena. To get at the extent and the source of non-ideally behaved water (an operational term dependent on the measurement method), we studied the motional and colligative properties of water in cells, in solutions of amino acids and glycine peptides whose surface characteristics are known, and in solution of bovine serum albumin, hemoglobin and some synthetic polypeptides. Solutions of individual amino acids with progressively larger hydrophobic side chains showed one perturbed water molecule (structured-slowed in motion) per nine square angstroms of hydrophobic surface area. Water molecules adjacent to hydrophobic surfaces form pentagonal structural arrays, as shown by X-ray diffraction studies, that are reported to be disrupted by heat, electric field, hydrostatic pressure and phosphorylation state. Hydrophilic amino acids demonstrated water destructuring (increased motion) that was attributed to dielectric realignment of dipolar water molecules in the electric field between charge groups. In solutions of proteins, several methods indicate the equivalent of 2-8 layers of structured water molecules extending beyond the protein surface, and we have recently demonstrated that induced protein conformational change modifies the extent of non-ideally behaved water. Water self-diffusion rate as measured in three different cell types was about half that of bulk water, indicating that most of the water in these cells was slower in motion than bulk water. In different cell types the extent of osmotically perturbed water ranged from less that half to almost all of the intracellular water. The assumption that essentially all intracellular water has ideal osmotic and motional behavior is not supported by the experimental findings. The non-ideally of cell water is an operational term. Therefore, the amount of non-ideally behaving water is dependent on the characteristics of water targeted, i.e. the measurement method, and a large fraction of it is explainable in mechanistic terms at a molecular level based on solute-solvent interactions.

Maintenance of ions, proteins and water in lens fiber cells before and after treatment with non-ionic detergents.

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks' balanced salt solution in either absence or presence of non-ionic detergents (0.2% Triton X-100 or 0.2% Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K+, Mg2+, Na+, Ca2+, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 degree C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K+, Mg2+, water and cytomatrix proteins.

Osmotic pressure method to measure salt induced folding/unfolding of bovine serum albumin.

A new approach has been developed to monitor protein folding by utilizing osmotic pressure and a range of salt concentrations in a well characterized protein, bovine serum albumin (BSA). It is hypothesized that both the 'effective' osmotic molecular weight, Ae, and the solute/solvent interaction parameter, I, in the empirical relation Msolvent/Msolute = (RT rho/Ae)1/pi + I [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is thought to depend directly upon the solvent accessible surface area (ASA). It is reasoned that larger solvent accessible surface area of an unfolded or denatured protein should perturb more water and produce larger I-values. Thus I-values allow calculation of a unfolded protein fraction, fua, due to changes in relative solvent accessible surface area. It has been observed that Ac decreases for filamentous, denatured proteins due to segmental motion of the molecule [2]. This allows calculation of unfolded protein fraction from the effective molecular weight, fum. Colloid osmotic pressure of BSA was measured in a range of salt concentrations at 25 degrees C, and pH = 7 (above the isoelectric point of BSA at pH = 5.4). Both S and I were used to monitor protein folding as the salt concentration was varied. In general, larger and variable I-values and smaller Ae were observed at salt concentrations less than 50 mmolal NaCl (Imax = 8.9), while constant I = 4.1 and Ae = 66,500 were observed above 50 mmolal NaCl. The two expressions for fractional unfolding (fua and fum) are in general agreement. Small differences in the parameters below 50 mmolal salt concentration are explained with well known shifts in the relative amounts of alpha-helix, beta-sheet and random coil in denatured BSA. The relative amounts of these shifts agree with predictions in the literature attributed to continuous BSA expansion rather than an 'all-or-none' conversion.

Solution nonideality related to solute molecular characteristics of amino acids.

By measuring the freezing-point depression for dilute, aqueous solutions of all water-soluble amino acids, we test the hypothesis that nonideality in aqueous solutions is due to solute-induced water structuring near hydrophobic surfaces and solute-induced water destructuring in the dipolar electric fields generated by the solute. Nonideality is expressed with a single solute/solvent interaction parameter I, calculated from experimental measure of delta T. A related parameter, I(n), gives a method of directly relating solute characteristics to solute-induced water structuring or destructuring. I(n)-values correlate directly with hydrophobic surface area and inversely with dipolar strength. By comparing the nonideality of amino acids with progressively larger hydrophobic side chains, structuring is shown to increase with hydrophobic surface area at a rate of one perturbed water molecule per 8.8 square angstroms, implying monolayer coverage. Destructuring is attributed to dielectric realignment as described by the Debye-Hückel theory, but with a constant separation of charges in the amino-carboxyl dipole. By using dimers and trimers of glycine and alanine, this destructuring is shown to increase with increasing dipole strength using increased separation of fixed dipolar charges. The capacity to predict nonideal solution behavior on the basis of amino acid characteristics will permit prediction of free energy of transfer to water, which may help predict the energetics of folding and unfolding of proteins based on the characteristics of constituent amino acids.

Correction for solute/solvent interaction extends accurate freezing point depression theory to high concentration range.

The authors describe empirical corrections to ideally dilute expressions for freezing point depression of aqueous solutions to arrive at new expressions accurate up to three molal concentration. The method assumes non-ideality is due primarily to solute/solvent interactions such that the correct free water mass Mwc is the mass of water in solution Mw minus I.M(s) where M(s) is the mass of solute and I an empirical solute/solvent interaction coefficient. The interaction coefficient is easily derived from the constant in the linear regression fit to the experimental plot of Mw/M(s) as a function of 1/delta T (inverse freezing point depression). The I-value, when substituted into the new thermodynamic expressions derived from the assumption of equivalent activity of water in solution and ice, provides accurate predictions of freezing point depression (+/- 0.05 degrees C) up to 2.5 molal concentration for all the test molecules evaluated; glucose, sucrose, glycerol and ethylene glycol. The concentration limit is the approximate monolayer water coverage limit for the solutes which suggests that direct solute/solute interactions are negligible below this limit. This is contrary to the view of many authors due to the common practice of including hydration forces (a soft potential added to the hard core atomic potential) in the interaction potential between solute particles. When this is recognized the two viewpoints are in fundamental agreement.

A study of the molecular sources of nonideal osmotic pressure of bovine serum albumin solutions as a function of pH.

The nonideal osmotic pressure of bovine serum albumin (BSA) solutions was studied extensively by Scatchard and colleagues. The extent of pH- and salt-dependent nonideality changes are large and unexplained. In 1992, Fullerton et al. derived new empirical expressions to describe solution nonideal colligative properties including osmotic pressure (Fullerton et al. 1992. Biochem. Cell Biol. 70:1325-1331). These expressions are based on the concepts of volume occupancy and hydration force. Nonideality is accurately described by a solute/solvent interaction parameter I and an "effective" osmotic molecular weight Ae. This paper uses the interaction-corrected nonideal expressions for osmotic pressure to calculate the hydration I values and "effective" osmotic molecular weight of BSA, Ae, as a function of pH. Both factors vary in a predictable manner due to denaturing of the BSA molecule. Both contribute to an increase in osmotic pressure for the same protein concentration as the solution pH moves away from the isoelectric point. Increased nonideality is caused by larger hydration resulting from larger solvent-accessible surface areas and by the decrease in "effective" osmotic molecular weight, Ae, due to segmental motion of denatured (filamentous) molecules.

Method to improve the accuracy of membrane osmometry measures of protein molecular weight.

Membrane osmometry provides a simple method to determine protein molecular weight but accuracy is limited by nonideal behavior. Recent studies (Fullerton et al., Biochem. Cell Biol., in press) show that non-ideal osmotic response of protein solutions is described by the empirical equation, Msv/M(s) = RT rho/A(s) x 1/II+I, where M(s) = mass of solute, Msv = mass of solvent, R = the Universal gas constant, T = absolute temperature, rho = solvent density, A(s) = solute molecular mass, II = osmotic pressure, and I = the nonideality parameter. This linear relation is used in this paper to demonstrate that measurement of molecular weight from the slope simplifies such measures and improves the accuracy relative to classical methods. The molecular weight of bovine serum albumin is measured with error less than 0.9%. The single dimensionless non-ideality parameter, I = 4.05 + 0.07, describes non-ideal curvature in the typical IIV = nRT diagram better than the customary second power viral expansion requiring 3 fitting constants. Analysis of eight data sets on four proteins from the literature shows that molecular weight calculated from the slope of the new equation agrees with chemical molecular weight within an RMS error of only 1.9%.

Solute/solvent interaction corrections account for non-ideal freezing point depression.

A new highly accurate curve-fitting technique for looking at freezing-point depression data was proposed by Fullerton et al. (Biochem. Cell Biol., in press). The method involve plotting mass solvent to mass solute ratio (Mw/M(s)) vs. 1/delta T (i.e. the inverse change in freezing point). A measured molecular weight and a solute/solvent interaction parameter (called I value) are inferred from the resultant linear plot. The accuracy of the molecular weight method was first demonstrated with the monomers of ethylene glycol, glycerol, propanol, mannitol, glucose and sucrose to show a mean molecular weight error of 0.02% with root mean square (RMS) error 0.9%. The RMS error (0.9%) is our best estimate of the molecular weight measurement accuracy for the method applied to a monomer. This error is consistent with the experimental precision (approximately 1%) which implies no systematic error. Non-ideality is described with a single constant, I. Polyethylene glycol (PEG) polymers of increasing length (vendor designation 200 to 10,000 Da) were analyzed to show monotonically increasing non-ideality (I values of 0.12 to 3.67) with increasing molecular weight. The measured molecular weights agreed with the end-point titration value for the three smallest polymers (where the number of polymeric units was less than or equal to 7). The method underestimates the vendor molecular weights for longer polymers. This disagreement is assigned to segmental motion (internal entropy) of longer, more flexible, PEG molecules.

Changes in the concentration of ions during senescence of the human erythrocyte.

Flash frozen samples of normal human blood were cryosectioned and cryodried for electron probe x-ray microanalysis of the concentration of ions and elements in individual erythrocytes (RBCs). The data (expressed in mM/kg dry weight) demonstrated a systematic pattern of variation between the concentration of ions and elements in the RBCs. Specifically as K+ and Cl- decreased in concentration, Ca2+ and sulfur increased in concentration. Phosphorous, Na+ and Mg2+ did not demonstrate a significant pattern of change. These findings are related to the dehydration and to the volume decrease that accompanies senescence of the RBC.

New expressions to describe solution nonideal osmotic pressure, freezing point depression, and vapor pressure.

New empirical expressions for osmotic pressure, freezing point depression, and vapor pressure are proposed based on the concepts of volume occupancy and (or) hydration force. These expressions are in general inverse relationships in comparison to the standard ideal expressions for the same properties. The slopes of the new equations are determined by the molecular weight of the solute and known constants. The accuracy and precision of the molecular weights calculated from the slope are identical and approximately 1% for the experiments reported here. The nonideality of all three colligative expressions is described by a dimensionless constant called the solute-solvent interaction parameter I. The results on sucrose have the same I = 0.26 for all three solution properties. The nonideality parameter I increased from 0.26 on sucrose to 1.7 on hemoglobin to successfully describe the well-known nonideal response of macromolecules.

Proton relaxation enhancement associated with iodinated contrast agents in MR imaging of the CNS.

PURPOSE: To study the effects of iodinated radiographic contrast agents on proton relaxation in MR imaging. PATIENTS AND METHODS: Two patients were evaluated after the intrathecal administration of an iodinated nonionic contrast agent (Isovue) and five subjects with cranial tumors following the intravenous administration of an iodinated ionic contrast medium (Renografin). RESULTS: Both patients with subarachnoid iodinated contrast media demonstrated a relative reduction in T1 and/or T2 times using a spin-echo sequence, while four of five of the subjects with intracranial tumors (one glioma, one dural metastasis, three meningiomas) and intravenous enhancement revealed a visible MR effect. Confirmation of these in vivo observations was obtained by in vitro measurement of T1 and T2 while varying the concentration of the contrast media in saline. All iodinated contrast media showed progressively reduced relaxation times (T1 and T2) as the concentration of the agent was increased. The largest contributing relaxation mechanism is probably due to the binding and exchange of the surrounding water with the contrast molecules. CONCLUSION: The observed T1/T2 effects suggest that administration of iodinated contrast media in the period immediately prior to MR scanning may be contraindicated in selected cases due to the demonstrated alteration of MR signal intensity that may lead to diagnostic inaccuracies.

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D2O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D2O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D2O saline or in a D2O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte "shells" revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.

Ion and water distribution in pig lenses incubated at 0 degree C to disable ion transport pumps.

This study was designed to test how extended exposure of lenses to sera with different ionic strengths influences the distribution of ions and water in the lens. Pig lenses were incubated in cold sera (0 degree C), which were adjusted to variable concentrations of NaCl, and their K+, Na+, Cl-, and water contents were measured. Incubation at 0 degree C inhibits active transport processes and thereby allows equilibration of the mobile ions and water. The hypothesis was that lens water content (volume) would follow the ion-induced protein changes predicted by a model derived from previous osmotic studies on proteins. As expected, exposure of the lens to cold caused a gain of sodium and a partial loss of potassium. However, the potassium concentration in the lens remained several fold higher than that in the bathing solution (about 41 vs. 1.8-4.6 mM/kg H2O), indicating that a portion of the potassium within the cold-exposed lens was not free to diffuse. That the water content of the lens showed a negative rather than a positive relationship with the concentration of NaCl within the lens was explained by the idea that an increase in NaCl within the lens (up to at least 250 mM/kg H2O) causes a decrease in the osmotically unresponsive water volume associated with lens proteins.