Transport properties of charged membranes undergoing conformational transitions

Summary The permeability and reflection behavior of cross-linked collagen films in dilute salt solutions have been investigated by measurements of net volume flow, isotopic exchange of THO and of Ca⁴⁵, and osmotic pressure. Complementary measurements of swelling, membrane resistance, membrane potential, and streaming potential are presented. Measurements were performed in the pH range of 5 to 1.5, at temperatures between 25 and 52 °C, and in the presence of KCl, 10^–2 m or CaCl₂, 10^–3 m. Under the conditions adopted, the membrane carries a net positive charge and undergoes large changes in degree of swelling (Donnan effect) and structure (crystal amorphous transition). The results indicate that when pH is lowered the filtration coefficientL _p decreases in the crystalline state (pH 5 to 3), increases during the conformational transition (pH 3 to 2), and decreases in the amorphous state (pH<2). It appears thatL _p is affected more by such properties as structure and porosity (i.e., mechanical resistance to flow) than properties related to the charged character of the membrane. The reflection coefficient increases when pH is lowered until pH 3, and decreases upon further lowering of pH. Such behavior is described in terms of the competition between swelling (due to both the Donnan effect and the melting transition) and fixed-charge density. Values of fixed-charge density derived on the basis of a theoretical expression for were found to be in good agreement with independent titration data.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : I. THE BEHAVIOR AND PROPERTIES OF COMMERCIAL COLLODION

1. The electrochemical behavior of membranes prepared from commercial collodion preparations varies widely, some preparations showing very high, other ones very low electrochemical efficiency ("activity"). 2. The electrochemical activity of a collodion membrane depends entirely upon impurities of an acidic nature contained in the collodion used for casting the membrane. 3. The active acidic impurities are substantially due to partial oxidation which occurs in the manufacturing process. Sulfuric acid compounds; e.g., acid sulfuric acid esters play only a minor rôle, if any. 4. The electrochemical behavior of collodion membranes in solutions of strong electrolytes is decisively dependent upon the acidic groups built permanently into the collodion surfaces. Preferential ion adsorption plays only a minor, if any, rôle.     

Estimation of surface charges in some biological membranes.

The resting membrane potential data existing in the literature for the giant axon of the squid, frog muscle and barnacle muscle have been analyzed from the standpoint of the theory of membrane potential due to Kobatake and co-workers. The average values derived for the effective charge density phi chi (where phi is a constant, 0 less than phi less than 1, and represents the fraction of counterions that are free, and chi is the stoichiometric charge density in the membrane) present on the different biomembranes existing in their normal ionic environment are 0.3, 0.325 and 0.17 M for the squid axon, frog and barnacle muscles, respectively. On the assumption that the values of phi are 0.4 and 0.2 for nerve and muscle membranes, respectively, values of 0.75, 1.62 and 0.85 M have been derived for the stoichiometric charge density (chi) present in the respective biological membranes. These correspond to 1 negative charge per 222, 103 and 195 A2 of the membrane area of the squid axon, frog and barnacle muscles, respectively.     

THE EFFECT OF pH ON THE PERMEABILITY OF COLLODION MEMBRANES COATED WITH PROTEIN

The permeability of gelatin-coated collodion membranes, as measured by the flow of water or of dilute solutions through the membranes, has been found to vary with the pH of the solutions. The permeability is greatest near the isoelectric point of the protein; with increasing concentration of either acid or alkali it decreases, passes through a minimum, and then increases. These variations with pH are qualitatively in accord with the assumption that they are due to swelling of the gelatin in the pores of the membrane, the effects of pH being similar to those observed by Loeb on the swelling of gelatin granules. Indications have been found of a similar variable permeability in the case of membranes coated with egg albumin, edestin, serum euglobulin, and serum albumin.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IX. WATER UPTAKE AND SWELLING OF COLLODION MEMBRANES IN AQUEOUS SOLUTIONS OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES

1. Dried collodion membranes are known to swell in water and to the same limited extent also in solutions of strong inorganic electrolytes (Carr and Sollner). The present investigation shows that in solutions of organic electrolytes and non-electrolytes, the swelling of dried collodion membranes is not as uniform, but depends on the nature of the solute. 2. The solutions of typically "hydrophilic" substances, e.g., glycerine, glucose, and citric acid, swell collodion membranes only to the same extent as water and solutions of strong electrolytes. In solutions of typically carbophilic substances (e.g., butyric acid, valeric acid, isobutyl alcohol, valeramide, phenol, and m-nitrophenol) the swelling of the membranes is much stronger than in water, according to the concentration used. For the brand of collodion used the swelling in 0.5 M solution was in some cases as high as 26 per cent of the original volume, as compared to 6 to 7 per cent in water. Therefore, in these solutions the "water-wetted dried" collodion membrane is not rigid, inert, and non-swelling, but behaves as a swelling membrane. 3. The solutes which cause an increased swelling of the membranes are accumulated in the latter, the degree of accumulation being markedly parallel with the degree of their specific swelling action. 4. The anomalously high permeabilities of certain carbophilic organic solutes reported by Michaelis, Collander, and Höber find an explanation in the specific interaction of these substances with collodion. 5. The use of the collodion membrane as a model of the ideal porous membrane is restricted to those instances in which no specific interaction occurs between the solute and the collodion.     

a-c electrical properties of bipolar membranes: Experiments and a model

Summary The a-c electrical properties of bipolar membranes separating equal strength solutions of the same uni-univalent electrolyte are analyzed for the case where both ions have equal mobilities. Two membrane models are treated. In one, the fixed-charge density is assumed to be constant throughout the membrane. In the other, the membrane is regarded as comprising an array of pores separated by walls through which the fixed charge is spread uniformly. Experimental results are reported for the a-c electrical properties of a bipolar membrane prepared from a single polyolephine sheet and immersed in KCl solutions of various concentrations. It is found that the data can be interpreted using the pore model.     

Effects of initial-fixed charge density on pH-sensitive hydrogels subjected to coupled pH and electric field stimuli: a meshless analysis

In this paper, we study the effects of initial fixed-charge density on the response behavior of pH-sensitive hydrogels subjected to coupled stimuli, namely, solution pH and externally applied electric field. This is the first instance in which a coupled stimuli numerical analysis has been carried out for these polymer gels, which are used as active sensing/actuating elements in advanced biomicroelectromechanical systems devices. In this work, a chemo-electro-mechanical formulation, termed the multi-effect-coupling pH-stimulus (MECpH) model, is first presented. This mathematical model takes into account the ionic species diffusion, electric potential coupling, and large mechanical deformation. In addition, a correlation between the diffusive hydrogen ions and fixed-charge groups on the hydrogel polymeric chains is established based on the Langmuir absorption isotherm, and incorporated accordingly into the MECpH model. To solve the resulting highly nonlinear and highly coupled partial differential equations of this mathematical model, the Hermite-Cloud method, a novel true meshless technique, is employed. To demonstrate the accuracy and robustness the MECpH model, computed numerical results are compared with experimental data available from literature. Following this validation, several numerical studies are carried out to investigate the effects of initial fixed-charge density on the volumetric variations of these pH-stimulus-responsive hydrogels when immersed in buffered solutions.     

Studies with Composite Membranes: I. Preparation and Measurement of Impedances

Simple and composite membranes have been prepared from 2% collodion solutions containing different amounts of polystyrenesulfonic acid (PSSA). Various membrane parameters such as water content, electrolyte uptake, exchange capacity, and permselectivity of these membranes have been determined. The resistance and capacitance of simple membranes have been measured as functions of both external electrolyte concentration and internal fixed charge density. The impedance characteristics of composite membranes also have been determined and discussed in terms of the resistance and capacitance characteristics of simple membranes from which the composite structures have been formed.     

Physical behavior of gelatin gels: a simple model for connective tissue

The swelling behavior of gelatin gels when immersed in buffer or in osmotically active solutions of dextran 500 has been studied, and the results can be described by means of the Flory-Rehner theory of the thermodynamic properties of three-dimensional network structures. The density of crosslinkages formed in the gel is found to be related to the concentration of gelatin at which gelation occurs. Values of rigidity moduli are estimated from the density of crosslinkages by use of the statistical thermodynamic theory of rubber elasticity. These values are in excellent agreement with direct measurements.     

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.     

THE KINETICS OF OSMOSIS

It is shown that by combining the osmotic pressure and rate of diffusion laws an equation can be derived for the kinetics of osmosis. The equation has been found to agree with experiments on the rate of osmosis for egg albumin and gelatin solutions with collodion membranes.     

THE ISOELECTRIC POINT OF A STANDARD GELATIN PREPARATION1

Two samples of a standard gelatin were studied, both prepared according to published specifications and washed free from diffusible electrolytes. The isoelectric point of this material was determined in four ways. 1. The pH values of solutions of gelatin in water approached the limit 4.86 ± 0.01 as the concentration of gelatin was increased. 2. The pH values of acetate buffers were unchanged by the addition of gelatin only at pH 4.85 ± 0.01. This gives the isoionic point of Sørensen, which is the isoelectric point with respect only to hydrogen and hydroxyl ions. 3. Gels of this gelatin made up in dilute HCl or NaOH, or in dilute acetate buffers, exhibited maximum turbidity at pH 4.85 ± 0.03. 4. Very dilute suspensions of collodion particles in 0.1 per cent gelatin solutions made up in acetate buffers showed zero velocity in cataphoresis experiments only at pH 4.80 ± 0.01. No evidence was found for the assumption that gelatin has two isoelectric points at widely separated pH values. It is concluded that the isoelectric point of this standard gelatin is not far from pH 4.85.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : V. THE INFLUENCE OF THE THICKNESS OF DRIED COLLODION MEMBRANES UPON THEIR ELECTROMOTIVE BEHAVIOR

1. Experiments were carried out to decide whether or not the electromotive properties of dried collodion membranes depend upon their thickness. 2. A number of dried collodion membranes of varying thickness, 3–160 µ, were prepared from collodion preparations of different electrochemical activity. The characteristic concentration potentials across them were measured and the means of these values determined for each thickness. 3. The characteristic concentration potentials across dried collodion membranes are a function of their thickness. The thinnest membranes yield in all cases the lowest concentration potentials; increasingly thicker membranes give increasingly higher potential values, until a constant value is reached which is characteristic of the particular collodion preparation used. With electrochemically active collodion, characteristic concentration potentials approaching the thermodynamically possible maximum are obtained with membranes of only 10 µ thickness, thinner membranes giving appreciably lower values. With two rather inactive commercial collodion preparations the characteristic concentration potential increases from about 30 mv. for membranes 3 µ thick to about 42 mv. for 20 µ membranes; still thicker membranes do not show a significant increase in the potential values. With a highly purified collodion preparation the constant maximum value was found to be about 32 mv., 4 µ thick membranes giving only about 22 mv. 4. These results do not support the homogeneous phase theory as applied to the dried collodion membrane. They are readily compatible with the micellar-structural theory. Several special possible cases of the latter as applied to the dried collodion membrane are discussed.     

Myoplasmic fixed charges of the barnacle muscle fiber (author's transl)]

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and phi X, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061--1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced phi X, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100 g KCl concentration in the external medium equal to 0.15 M, phi X was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in phi X of 20--50% depending on E1KCl] in the external solutions. The addition of ATP, Mg2+, and Ca2+ also causes a reduction of phi B by 30--50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when CKl is replaced by Nal. It also suggests a reduction of phi X during muscular contraction.     

Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes

1. We measured changes in resting membrane potential (Em) and Na-K pump activity, assayed by ouabain-sensitive 86Rb uptake, in response to carbamylcholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control Em (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of Em was most apparent by culture age 8-9 days in vitro (DIV), Em reaching values as high as -60 mV by 5-10 min after peak depolarization at 37 degrees C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of Em and, when given after repolarization had begun, reversed it and caused Em to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which Em did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose Em repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in 86Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of Em following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.     

THE ELIMINATION OF DISCREPANCIES BETWEEN OBSERVED AND CALCULATED P.D. OF PROTEIN SOLUTIONS NEAR THE ISOELECTRIC POINT WITH THE AID OF BUFFER SOLUTIONS

1. It had been noticed in the previous experiments on the influence of the hydrogen ion concentration on the P.D. between protein solutions inside a collodion bag and aqueous solutions free from protein that the agreement between the observed values and the values calculated on the basis of Donnan''s theory was not satisfactory near the isoelectric point of the protein solution. It was suspected that this was due to the uncertainty in the measurements of the pH of the outside aqueous solution near the isoelectric point. This turned out to be correct, since it is shown in this paper that the discrepancy disappears when both the inside and outside solutions contain a buffer salt. 2. This removes the last discrepancy between the observed P.D. and the P. D. calculated on the basis of Donnan''s theory of P.D. between membrane equilibria, so that we can state that the P.D. between protein solutions inside collodion bags and outside aqueous solutions free from protein can be calculated from differences in the hydrogen ion concentration on the opposite sides of the membrane, in agreement with Donnan''s formula.     

Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata.

The midpoint potentials of the mitochondrial respiratory chain cytochromes of the protozoan Crithidia fasciculata at pH 7.2, Em7.2, show great similarity to those measured in higher organisms. Values of Em7.2 for cytochromes a and a3 are +165 and +340 mV. Both c cytochromes have Em7.2 = +230 mV. There are two b cytochromes with the same spectral characteristics with Em7.2 = -20 and -135 mV. These values are compatible with two sites of energy conservation for oxidative phosphorylation in these mitochondria. All cytochrome components show potentiometric titrations with n = 1. There is a fluorescent flavoprotein in these mitochondria with Em7.2 = -40 mV and n =2, whose function is not known.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IV. THE RELATIVE MERITS OF THE HOMOGENEOUS PHASE THEORY AND THE MICELLAR-STRUCTURAL THEORY AS APPLIED TO THE DRIED COLLODION MEMBRANE

1. Experiments were carried out to decide whether a homogeneous phase (solubility) theory or a micellar-structural theory more adequately describes the behavior of dried collodion membranes with solutions of strong electrolytes. 2. A number of dried collodion membranes were prepared from an electrochemically inactive collodion preparation (state I); the characteristic concentration potentials across them were low, about 30 mv. The membranes were activated by oxidation (state II) to give maximum or nearly maximum concentration potentials (about 50 mv.). The oxidized membranes are dried, dissolved in alcohol-ether, and a new set of dry collodion membranes prepared from this solution (state III). The concentration potentials across these membranes are low. 3. Since the properties of a homogeneous phase should not be influenced by a rearrangement of its constituent particles, the experimental results do not support a homogeneous phase (solubility) theory, but they agree with the predictions of the micellar-structural theory. The characteristic behavior of dried collodion membranes in solutions of strong inorganic electrolytes is therefore due to the micellar character of its interstices.