The free-solvent model has been shown to have excellent predictability of the osmotic pressure for single and binary non-interactive proteins in aqueous solutions. Here the free-solvent model is extended to be more generalized by including the contributions of intra- and inter-protein interactions to the osmotic pressure of a solution in the form of homo- and hetero-multimers. The solute-solvent interactions are considered to be unique for each homo- and hetero-multimer in solution. The effect of the various generalized free-solvent model parameters on the osmotic pressure are examined for a single protein solution with a homo-dimer, a binary protein solution with no protein-protein interactions, and a binary protein solution with a hetero-dimer. Finally, the limitations associated with the generalized free-solvent model are discussed.

A negative second virial coefficient has long been a predictor of potential protein crystallization and salting out. However, the assumption that this is due to attractive solute-solute interactions remains a source of debate. Here we reexamine the second virial coefficient from protein osmometry in terms of the free-solvent model. The free-solvent model has been shown to provide excellent predictions of the osmotic pressure of concentrated and crowded environments for aqueous protein solutions in moderate ionic strengths. The free-solvent model relies on two critical parameters, hydration and ion binding, both which can be determined independently of osmotic pressure data. Herein, the free-solvent model is mathematically represented as a virial expansion model and the second virial coefficient is expressed in terms of solute-solvent interactions, namely hydration and ion binding. Hydration and ion binding values are then used to estimate the second virial coefficient at various protein concentrations for three model proteins ovalbumin (OVA), bovine serum albumin (BSA), and hen egg lysozyme (HEL) in various monovalent salt aqueous solutions. The results show that the conditions for obtaining a negative second virial coefficient emerge when the ionic strength of the influenced region of the protein is higher than that of the bulk. This analysis suggests a plausible explanation as to why proteins are more favorable for salting out or crystallization when the solution is represented by a negative second virial coefficient.

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPx(r), as expected, but is a function of both GPx(r) and GSH when the initial GPx(r) is less than H2O2. This simulation is supported by the biological observations of Li et al. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity x GSH (effective GPx activity). The predicted cellular average GPx(r) and H2O2 for their study are approximately GPx(r) < or =1 microm and H2O2 approximately 5 microm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.

The membrane concentration osmometer coupled with multiple sample preparations has been used for over a century to determine a number of colloidal properties. At the dilute region, this method has been used to determine solute molecular mass. When the solution is proteinaceous, in the intermediate region, the osmotic pressure profile provides the second virial coefficient, useful for estimating protein crystallization and salting out. At the most crowded concentrations, it provides insight into protein hydration and protein-ion interaction. One of the most critical factors in generating the osmotic pressure profile is minimizing the quantity of protein used and reducing the error in preparing samples. Here, we introduce a membrane concentrating osmometer that allows one to measure osmotic pressure over a wide concentration range from a single sample. A test study was performed using the osmotic pressure profile of self-crowded bovine serum albumin solutions. The resulting profile was in good agreement with previous data in the literature obtained from multiple sample studies. The osmotic pressure profile was further used with a free solvent-based osmotic pressure model to determine protein hydration and ion binding. These results were in excellent agreement with literature values. This concentrating osmometer has several advantages over a conventional concentration osmometer for obtaining the osmotic pressure profile for proteinaceous solutions: (1) the amount of protein required is significantly decreased, (2) the potential for experimental error in sample preparation diminishes, and (3) the time for generating the osmotic pressure profile is substantially reduced.

Osmotic transport devices (OTDs) are forward osmosis membrane devices that we recently developed to remove accumulated fluid from swollen tissue, in-vivo, under severe conditions. As such, the relative volume of the fluid required to be removed and the volumetric flowrate may be two orders of magnitude less than the operating volume and tangential flowrate of the device. This makes it challenging to measure the rate of fluid flow from the swollen tissue. Here, we introduce a differential densimetry method for determining ultra-low volumetric flux through tissue samples. This technique uses two vibrating tube density sensors, one placed upstream of the membrane in contact with the tissue sample, and one placed downstream. Any flow of biological fluid withdrawn through the tissue will combine with the OTD operating fluid resulting in an observed density shift in the second density sensor. By measuring the difference in density between the upstream and downstream fluids, one can calculate the amount of fluid flowing across the tissue with a relatively high level of sensitivity. This method is also relatively insensitive to drift from temperature fluctuations and capable of continuously monitoring tissue permeability in real time. As a proof of concept, we used this technique to measure fluid flow across ex-vivo rat spinal tissue for an appropriately scaled OTD. The repeatability error had a maximum of only 12%. This implies that this method can provide highly acceptable flux measurements with reasonable reproducibility in real-time applications of fluid removal in-vivo.