Effect of protein adsorption on the transport characteristics of asymmetric ultrafiltration membranes.

The effects of bovine serum albumin adsorption on the transport characteristics of asymmetric poly(ether sulfone) ultrafiltration membranes were determined using polydisperse dextrans with gel permeation chromatography. Actual dextran sieving coefficients were evaluated from observed sieving data for both the clean and preadsorbed membranes using a stagnant film model. The flux dependence of the actual dextran sieving coefficients was used to evaluate the intrinsic membrane hindrance factors for convective (i.e., sieving) and diffusive transport for the different molecular weight dextrans using classical membrane transport theory. Protein adsorption caused a reduction in both dextran sieving and diffusion, with the magnitude of the reduction a function of the dextran molecular weight and pore size. The effects of adsorption on the specific pore area and the membrane porosity were then determined using a recent model for solute transport through asymmetric ultrafiltration membranes. The data indicate that protein adsorption occurs preferentially in the larger membrane pores, causing a greater reduction in solute sieving compared to the membrane hydraulic permeability and porosity than would be predicted on the basis of either a simple pore blockage or pore constriction model.     

Coupling of Solute and Solvent Flows in Porous Lipid Bilayer Membranes

The present experiments were designed to evaluate coupling of water and nonelectrolyte flows in porous lipid bilayer membranes (i.e., in the presence of amphotericin B) in series with unstirred layers. Alterations in solute flux during osmosis, with respect to the flux in the absence of net water flow, could be related to two factors: first, changes in the diffusional component of solute flux referable to variations in solute concentrations at the membrane interfaces produced by osmotic flow through the unstirred layers; and second, coupling of solute and solvent flows within the membrane phase. Osmotic water flow in the same direction as solute flow increased substantially the net fluxes of glycerol and erythritol through the membranes, while osmotic flow in the opposite direction to glycerol flow reduced the net flux of that solute. The observed effects of osmotic water flow on the fluxes of these solutes were in reasonable agreement with predictions based on a model for coupling of solute and solvent flows within the membrane phase, and considerably in excess of the prediction for a diffusion process alone.     

A mathematical model of solute coupled water transport in toad intestine incorporating recirculation of the actively transported solute

A mathematical model of an absorbing leaky epithelium is developed for analysis of solute coupled water transport. The non-charged driving solute diffuses into cells and is pumped from cells into the lateral intercellular space (lis). All membranes contain water channels with the solute passing those of tight junction and interspace basement membrane by convection-diffusion. With solute permeability of paracellular pathway large relative to paracellular water flow, the paracellular flux ratio of the solute (influx/outflux) is small (2-4) in agreement with experiments. The virtual solute concentration of fluid emerging from lis is then significantly larger than the concentration in lis. Thus, in absence of external driving forces the model generates isotonic transport provided a component of the solute flux emerging downstream lis is taken up by cells through the serosal membrane and pumped back into lis, i.e., the solute would have to be recirculated. With input variables from toad intestine (Nedergaard, S., E.H. Larsen, and H.H. Ussing, J. Membr. Biol. 168:241-251), computations predict that 60-80% of the pumped flux stems from serosal bath in agreement with the experimental estimate of the recirculation flux. Robust solutions are obtained with realistic concentrations and pressures of lis, and with the following features. Rate of fluid absorption is governed by the solute permeability of mucosal membrane. Maximum fluid flow is governed by density of pumps on lis-membranes. Energetic efficiency increases with hydraulic conductance of the pathway carrying water from mucosal solution into lis. Uphill water transport is accomplished, but with high hydraulic conductance of cell membranes strength of transport is obscured by water flow through cells. Anomalous solvent drag occurs when back flux of water through cells exceeds inward water flux between cells. Molecules moving along the paracellular pathway are driven by a translateral flow of water, i.e., the model generates pseudo-solvent drag. The associated flux-ratio equation is derived.     

Transport across homoporous and heteroporous membranes in nonideal, nondilute solutions. II. Inequality of phenomenological and tracer solute permeabilities.

The phenomenological solute permeability (omega p) of a membrane measures the flux of solute across it when the concentrations of the solutions on the two sides of the membrane differ. The relationship between omega p and the the conventionally measured tracer permeability (omega T) is examined for homoporous and heteroporous (parallel path) membranes in nonideal, nondilute solutions and in the presence of boundary layers. In general, omega p and omega T are not equal; therefore, predictions of transmembrane solute flux based on omega T are always subject to error. For a homoporous membrane, the two permeabilities become equal as the solutions become ideal and dilute. For heteroporous membranes, omega p is always greater than omega T. An upper bound on omega p- omega T is derived to provide an estimate of the maximum error in predicted solute flux. This bound is also used to show that the difference between omega P and omega T demonstrated earlier for the sucrose-Cuprophan system can be explained if the membrane is heteroporous. The expressions for omega P developed here support the use of a modified osmotic driving force to describe membrane transport in nonideal, nondilute solutions.     

Effect of heteroporosity on flux equations for membranes

An investigation is made of the possible errors in simple integrated equations for solute flux across both non-pieving and sieving porous membranes that can result from variations in the membrane structure. Detailed structural models are used, beginning with a membrane consisting of a parallel array of pores and progressing to series-parallel combinations of pore segments of various lengths and cross-sectional areas, with internal cross connections among pore segments allowed. It is shown that there are both upper and lower mathematical bounds on the possible variations that can be produced in a curve of solute flux versus volume flow by arbitrary variation in the membrane structure, subject only to certain general conditions. In particular, the flux equation for a homoporous membrane is a lower bound- The maximum deviations from this lower bound for a membrane of arbitrary structure are only moderately large, and require rather extreme pore size distributions; most distributions introduce only small errors. Implications of these results in studies of real membrane structure and in the design of experiments are discussed.     

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

Effects of solvent and solute drag on transmembrane diffusion

The present study compares and quantitates both solvent drag and solute drag forces in a system with both heteropore and homopore membranes. It is shown that tracer solute permeability can be increased if solution flow or driver solute flux is in the direction of tracer diffusion. Either force can decrease tracer permeability if the force can decrease tracer permeability if the force is opposite to the direction of tracer diffusion. The two forces can be additive or one force may reduce the effect of the other force. In the particular system quantitated, solute drag is shown to be some 300 times more effective than solvent drag on a mole-to-mole basis. The use of a number of solute pairs on other homopore and heteropore membranes confirms the finding that the two drag forces can be analyzed or manipulated in a variety of systems.     

Responding phospholipid membranes--interplay between hydration and permeability.

Osmotic forces are important in regulating a number of physiological membrane processes. The effect of osmotic pressure on lipid phase behavior is of utmost importance for the extracellular lipids in stratum corneum (the outer part of human skin), due to the large gradient in water chemical potential between the water-rich tissue on the inside, and the relative dry environment on the outside of the body. We present a theoretical model for molecular diffusional transport over an oriented stack of two-component lipid bilayers in the presence of a gradient in osmotic pressure. This gradient serves as the driving force for diffusional motion of water. It also causes a gradient in swelling and phase transformations, which profoundly affect the molecular environment and thus the local diffusion properties. This feedback mechanism generates a nonlinear transport behavior, which we illustrate by calculations of the flux of water and solute (nicotine) through the bilayer stack. The calculated water flux shows qualitative agreement with experimental findings for water flux through stratum corneum. We also present a physical basis for the occlusion effect. Phase behavior of binary phospholipid mixtures at varying osmotic pressures is modeled from the known interlamellar forces and the regular solution theory. A first-order phase transformation from a gel to a liquid--crystalline phase can be induced by an increase in the osmotic pressure. In the bilayer stack, a transition can be induced along the gradient. The boundary conditions in water chemical potential can thus act as a switch for the membrane permeability.     

Simulation study of interaction mechanism between peptide and asymmetric membrane

Cell-penetrating peptides (CPPs) are widely used as drug carriers, owing to their superior ability to cross cell membrane both alone and with cargos, such as genes and other particles. Understanding the translocation mechanism of CPP is significant for many therapeutic purposes, including targeting drug and gene delivery. In this study, we performed a coarse-grained molecular dynamics simulation to investigate the interaction mechanism between polyarginine peptides and asymmetric membranes. Results showed that peptides can penetrate through the lipid bilayer by inducing a hydrophilic hole formation in the asymmetric membrane. Furthermore, the lengthy peptide chain length (R4–R16 peptides) and high membrane asymmetry positively affect peptide penetration. Our study provides insights into the molecular-level interactions between peptides and asymmetric membranes, as well as suggestions for targeted gene and drug delivery.     

Experimental study of osmosis through a collodion membrane

Experiments were carried out on a collodion membrane in order to study the factors that determine direction and magnitude of net flow of water across a membrane permeable to the solvent and to some of the solutes present. The solutes used were all non-ionic. When only one solute was present and there was no difference of hydrostatic pressure across the membrane, water flowed toward the side where its vapor pressure was lower, but the rate of transfer depended upon the nature of the solute: for a given difference in osmolality across the membrane, the rate increased with the molecular volume of the solute and reached its maximum with the solute to which the membrane was impermeable. These results led to the experimental demonstration that in the presence of two or more solutes of different molecular volumes, of which one at least can diffuse through the barrier, the net transfer of water can take place against its vapor pressure gradient. Some of the physicochemical and physiological implications of the data are discussed.     

Fabrication and electromechanical characterization of freestanding asymmetric membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.     

Bipartite expressions for diffusional mass transport in biomembranes

Analytical expressions for solute diffusion through a membrane barrier for different initial and boundary conditions are available in the literature. The three commonest initial and boundary conditions are for a membrane without solute respectively immersed in a solution of constant concentration, immersed in such a solution for one side but with the other side isolated, and immersed in such a solution for one side and with the other side kept at zero concentration. The physical quantities for the first two initial and boundary conditions are concentration and average concentration (the total solute entering the membrane) with amperometric current (flux) and solute that permeates through the membrane (charge passed) for the third initial and boundary condition. Expressions for these methods in the literature are inconvenient for practical applications because of the infinite mathematical series required. An investigation of convergence of these expressions was therefore carried out. Simple but accurate bipartite expressions for these methods were constructed and provided theoretical support for studies on mass transport characterization of biomembranes. As a specific application, these expressions enabled a direct fit of the simulated observables to experimental values to obtain diffusion coefficients. For these initial and boundary conditions and corresponding physical quantities, simple one point methods for diffusion coefficient estimation are also suggested. These latter diffusion coefficients can be initial values for numerical fit methods.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Solute Flux Coupling in a Homopore Membrane

Our previous studies on solute drag on frog skin and synthetic heteropore membranes have been extended to a synthetic homopore membrane. The 150-Å radius pores of this membrane are formed by irradiation and etching of polycarbonate films. The membrane is 6-µm thick and it has 6 x 10⁸ pores cm^–2. In this study, sucrose has been used as the driver solute with bulk flow blocked by hydrostatic pressure. As before on heteroporous membranes, the transmembrane asymmetry of tracer solute is dependent on the concentration of the driver solute. Tracer sucrose shows no solute drag while maltotriose shows appreciable solute drag at 1.5 M sucrose. With tracer inulin and dextran, solute drag is detectable at 0.5 M sucrose. These results are in keeping with the previous findings on heteropore membranes. Transmembrane solute drag is the result of kinetic and frictional interaction of the driver and tracer solutes as the driver flows down its concentration gradient. The magnitude of the tracer flux asymmetry is also dependent on the size of the transmembrane pores.     

Self-cleaning membranes for ultrafiltration

In order to reduce the severe flux losses encountered during ultrafiltration of protein solutions, proteases were immobilized on Ultrafiltration membranes to hydrolyze the deposited solute molecules. Over a standard 22 hr run 25 to 78% improvement in cumulative permeate yield was obtained when processing 0.5% albumin or hemoglobin. It was also demonstrated that the flux enhancements were due to the biochemical action of the absorbed protease and not to its physical effect as a prefilter coat. with the aid of a model retardation of gel formation mechanism was demonstrated. Economics of the system were shown to be favorable, improving the rate of return on capital investment up to 50% by reduction of the total membrane area of the plant.     

Flux measurement in single cells by fluorescence microphotolysis

Fluorescence microphotolysis — widely employed for diffusion studies — can be used to measure transfer (flux) of fluorescent solutes through membranes in single cells and organelles. This article analyses the methodological basis of flux measurements, provides experimental tests, and discusses potential applications. The principle of the method is to equilibrate cells, organelles or vesicles with a fluorescent solute, to deplete the interior of individual cells etc. of fluorescene by the pulse of a high-intensity microbeam, and to monitor influx of solute by microfluorometry. Simple equations are given and a computer curve fitting program is described by which rate constants of influx and membrane permeability coefficients can be derived from fluorescence measurements. The permeability of individual leaky human erythrocyte ghosts to fluorescein-isothiocyanate-labelled bovine serum albumin has been measured under various conditions. Multiple exposure to the high-intensity microbeam had no effect on permeability within experimental error. Flux measurements have been also performed on individual vesicles of 1–2 m radius which had been derived from ghosts. The potential application of the method to sub-lightmicroscopic vesicles and to organelles within living cells is discussed.Abbreviation FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin