Solutions Of Physiological Ionic Strength Research Articles

Osmotic pressure enables facile high yield assembly of cell-like giant unilamellar vesicles in solutions of physiological ionic strength.

Giant unilamellar vesicles (GUVs) are single-walled, closed phospholipid bilayers that are useful as cellular mimics for synthetic biology and drug delivery. The assembly of GUVs in solutions of ionic concentrations >150 mM Na/KCl (salty solutions) is challenging. Here, we investigate the effects of temperature, chemical identity of small molecule and macromolecular polymers on the molar yield of free-floating GUVs in salty solutions using high resolution confocal microscopy and large dataset image analysis. We find that low gelling temperature agarose is the singular compound that produces free-floating GUVs at molar yields >10 % in salty solutions. All other macromolecular polymers, such as polyvinyl alcohol (PVA), fructose, and ultralow gelling temperature agarose, produced yields of GUVs of <7 %. Surface images show that for these other compounds, the formation of large numbers of lipid-polymer pseudobuds due to polymer dewetting reduces yields of GUVs. We propose a free-energy model of GUV assembly to explain the effects of polymers on the assembly of GUVs. Modulating the adhesion potential by varying ion concentration and valency yielded experimental data that supports our model's prediction - the free-energy for bud formation can be reduced by a contribution to the osmotic pressure of dissolving polymers. These results provide a quantitative comparison between established and novel methodology, as well as experimental and theoretical framework to guide studies for obtaining GUVs in physiological salt solutions.

Pushing the Boundaries of Interfacial Sensitivity in Graphene FET Sensors: Polyelectrolyte Multilayers Strongly Increase the Debye Screening Length

Nanomaterial-based FET sensors represent an attractive platform for ultrasensitive, real-time, and label-free detection of chemical and biological species. Nevertheless, because their response is screened by mobile ions, it remains a challenge to use them to sense in physiological ionic strength solutions. In this work, it is demonstrated, both experimentally and theoretically, that polyelectrolyte multilayers are capable of increasing the sensing range of graphene-based FETs. Potential shifts at graphene surfaces and film thickness are recorded upon the construction of PDADMAC/PSS polyelectrolyte multilayer (PEM) films. By correlation of the potential shift with the film thickness, the electrostatic screening length and the concentration of mobile ion inside the films have been deduced. Across the polymer interface the Debye length is increased more than 1 order of magnitude. The fundamentals of this strategy are described by a conceptually simple thermodynamic model, which accounts for the entropy loss ...

A nanoelectronic enzyme-linked immunosorbent assay for detection of proteins in physiological solutions.

Semiconducting nanowires are promising ultrasensitive, label-free sensors for small molecules, DNA, proteins, and cellular function. Nanowire field-effect transistors (FETs) function by sensing the charge of a bound molecule. However, solutions of physiological ionic strength compromise the detection of specific binding events due to ionic (Debye) screening. A general solution to this limitation with the development of a hybrid nanoelectronic enzyme-linked immunosorbent assay (ne-ELISA) that combines the power of enzymatic conversion of a bound substrate with electronic detection is demonstrated. This novel configuration produces a local enzyme-mediated pH change proportional to the bound ligand concentration. It is shown that nanowire FETs configured as pH sensors can be used for the quantitative detection of interleukin-2 in physiologically buffered solution at concentrations as low as 1.6 pg mL(-1). By successfully bypassing the Debye screening inherent in physiological fluids, the ne-ELISA promises wide applicability for ligand detection in a range of relevant solutions.

Myosin filament depolymerizes in a low ionic strength solution containing l-histidine

Myosin, one of the major myofibrillar proteins, forms a filamentous polymer and is insoluble in physiological and low ionic strength solutions. We have shown that myosin is soluble in a low ionic strength solution containing L-histidine. In this study, to clarify the role of L-histidine in the solubilization of myosin, we investigated effects of L-histidine on the filament formation and the morphology of myosin at a low ionic strength. In the presence of L-histidine, myosin formed a filamentous polymer in a physiological ionic strength solution and dispersed in a low ionic strength solution. Transmission electron microscopy showed that light meromyosin (LMM), the rod region of myosin, in a low ionic strength solution containing L-histidine was longer than that in a high ionic strength solution without L-histidine. L-histidine causes the elongation of LMM region of myosin contributing to the weakening of the myosin filament and the dissociation of myosin in a low ionic strength solution.

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary, to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).

Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes.

We have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mM NaCL as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitro aerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (1-3 microns). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.

The effects of temperature, pH, and magnesium on the diffusion coefficient of ATP in solutions of physiological ionic strength

Intracellular diffusive transport of adenosine triphosphate (ATP) is critical to cellular metabolism. Physical models predict that diffusion coefficients ( D) of small molecules are functions of temperature and viscosity of the diffusive environment. Therefore, changes in body temperature, commonly experienced by poikilotherms, are expected to result in changes in the rate of intracellular ATP transport. However, it has been postulated that changes in the electrical charge of ATP may influence the interaction between ATP and the cytosol and that the temperature sensitivity of D ATP may deviate from the predicted relationship. To investigate the effects of changes in electrical charge on the temperature sensitivity of D ATP, we measured D ATP under various conditions of temperature, pH, and pMg 2+. Changes in pH and pMg 2+ were used to alter the net charge of ATP, and D ATP was measured in solutions of physiological ionic strength. Results showed a positive correlation between D ATP and temperature; D ATP = 1.75 ± 0.09, 3.68 ± 0.14, and 4.64 ± 0.13 (mean ± S.E.M.) × 10 −6 cm 2/s at 5°C, 25°C, and 40°C, respectively. Changes in pH and pMg 2+ did not significantly influence D ATP, and the change in D ATP with respect to temperature was similar to that predicted on the basis of changes in temperature and viscosity of the aqueous medium.

Solubility of Chicken Breast Muscle Proteins in Solutions of Low Ionic Strength

Essentially all of the proteins of chicken breast muscle were soluble in sodium chloride solutions of physiological ionic strength or less and neutral pH. However, there was a critical order of treatment necessary to accomplish this. After removal of those proteins solubilized by homogenizing the tissue 1:10 (w/v) in water, it was necessary to solubilize a fraction of the remaining proteins in moderate concentrations (25−150 mM) of sodium chloride at neutral pH before the remaining proteins could be solubilized in water. Solubilization of chicken breast muscle proteins in water could be prevented or reversed by salt solutions of low concentration, suggesting that most of the solubility was not directly caused by proteolysis. SDS−polyacrylamide gel electrophoresis indicated that removal of specific peptides by moderate concentrations of sodium chloride at neutral pH was correlated with removal of the restriction on the water solubility of the remaining proteins. Keywords: Protein solubility; solubility of ...

A RE‐EXAMINATION OF MUSCLE PROTEIN SOLUBILITY

ABSTRACTMuscle proteins are conveniently characterized by their solubility properties. Sarcoplasmic proteins are defined as those soluble when muscle is extracted with water or solutions of low ionic strength, i.e., physiological or less. Myofibrillar proteins are generally thought to be soluble at elevated ionic strengths (&gt; 0.3). Close examination of the extractability and solubility properties of these two groups of proteins, indicates that their solubility properties are not as different as is generally believed. With both groups of proteins, distinctions must be made between extractability and solubility. Our research results have revealed that the proteins of eight different white muscles are essentially completely soluble in solutions of physiological ionic strength or less and neutral pH. Six of these muscles were from white fleshed fish, and one each was from a fatty fish and chicken breast muscle. There were differences among the muscles of the various species. In mackerel and chicken breast muscle, it appeared that certain proteins had to be removed before the remaining myofibrillar proteins could be solubilized in water.

Effects of low ionic strength media on passive human red cell monovalent cation transport.

1. The effect of low ionic strength media on the residual, i.e. (ouabain + bumetanide + Ca2+)-insensitive, K+ influx was characterized in human red blood cells. 2. This K+ flux was enhanced significantly in isotonic solutions of low ionic strength using sucrose to maintain constant osmolarity. This effect was found for fresh red blood cells as well as for stored (bank) red blood cells. However, the absolute magnitude of K+ influx in solutions of low ionic strength was halved for stored red blood cells. 3. Anion replacement of Cl- by CH3SO4- did not affect residual K+ fluxes, showing that Cl- -dependent transport pathways (e.g. the KCl co-transporter) are not involved in the low ionic strength effect. 4. The enhanced K+ influx in low ionic strength media was reversible when the cells were resuspended in a solution of physiological ionic strength. 5. K+ influx measured in light and dense fractions of erythrocytes (separated by centrifugation and corresponding to samples enriched with either 'young' or 'mature' red cells) showed that the low ionic strength effect does not change markedly with cell age. 6. Low ionic strength media elevated residual, i.e. (ouabain + bumetanide + Ca2+)-insensitive, influx of both K+ and Na+ by about the same amount. In both cases the flux was linear with concentration in the range investigated (0.25-10 mM). No significant increase in the uptake of the cations Ca2+ and lysine in low ionic strength solutions could be found. 7. In CH3SO4- -containing solutions of physiological ionic strength the residual K+ influx was almost independent of cell volume, whereas this flux in CH3SO4- -containing solutions of low ionic strength declined as cell volume was increased. 8. K+ flux measurements in solutions of different external pH, where NaCl was replaced by sodium gluconate or sodium glucuronate, showed that the reduced ionic strength is of more importance for the enhanced residual K+ influx than the changed transmembrane potential or the changed intracellular pH. However, a small pH dependence could be found, the K+ flux passing through a minimum around pHi 7.3. 9. Hydrostatic pressure enhanced the residual K+ flux in media of low ionic strength synergistically, so that very large fluxes (greater than 10 mmol (1 cells)-1 h-1) were obtained at 40 MPa. The apparent activation volumes (delta V*) for the pressure-sensitive K+ flux were -108 and -69 ml mol-1 in low ionic strength or physiological ionic strength solutions respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin.

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.

Covalent cross-linking of single fibers from rabbit psoas increases oscillatory power

Single fibers from chemically skinned rabbit psoas muscle were treated with 1-ethyl-3-[3-dimethyl-amino)proyl]-carbodiimide (EDC) at 20 degrees C after rigor was induced. A 22-min treatment resulted in 18% covalent cross-linking between myosin heads and the thin filament as determined by stiffness measurements. This treatment also results in covalent cross-linking among rod portions of myosin molecules in the backbone of the thick filament. The fibers thus prepared are stable and do not dissolve in solutions at ionic strengths as high as 1,000 mM. The preparation was subjected to sinusoidal analysis, and the resulting complex modulus data were analyzed in terms of three exponential processes, (A), (B), and (C). Oscillatory work (process B) was much greater in the cross-linked fibers than in untreated ones in activating solutions of physiological ionic strength (200 mM); this difference was attributed to the decline of process (A) with EDC treatment. Consequently, the Nyquist plot of the EDC-treated preparation exhibited an insect-type response. We conclude that, under these conditions, both cross-linked and non-cross-linked myosin heads contribute to the production of oscillatory power. The cross-linked preparations also exhibited oscillatory work in high ionic strength (500-1,000 mM) solutions, indicating that cross-linked myosin heads are capable of utilizing ATP to produce work. We conclude that process (A) does not relate to an elementary step in a cross-bridge cycle, but it may relate to dynamics outside the cross-bridge such as filament sliding or sarcomere rearrangement.

Physico-Chemical studies of the activity of urease and the development of a conductimetric urea sensor

Abstract The hydration-dependence of the hydrolysis of urea by urease (EC 3.5.1.5) has been studied using d.c. conductivity, a.c. dielectric, hydration isotherm, mass-spectrometric and proton injection techniques. It is shown that the enzymic activity of urease increases rapidly for hydrations greater than 6 wt.% and that the supply of protons to the enzyme is the rate controlling factor. These results support the concept proposed by Careri that the critical hydration for the onset of activity of some enzymes corresponds to the formation of a network of percolation pathways on the enzyme molecule's surface. The hydrolysis of urea by immobilised urease can be monitored directly as a result of the increased dielectric polarisability associated with the generation of electroactive ammonium ions within the immoblised enzyme structure. This has led to the development of a conductimetric sensor capable of detecting 0.2 mM urea in salt solutions of physiological ionic strength.

Unusual extraction of α-actinin from the adductor muscles of bivalve molluscs

1. 1. In contrast to rabbit skeletal muscle α-actinin, which is extracted only by low ionic strength solutions, scallop α-actinin has been extracted in a wide range of ionic strength and its relative content is maximal in physiological ionic strength solutions. 2. 2. The yield of α-actinin from scallop myofibrils sharply increases in the presence of NaHCO 3 which may be used for isolation of α-actinin from molluscs muscles. 3. 3. NaHCO 3-solution completely removes α-actinin from scallop myofibrils, although α-actinin from rabbit skeletal muscles has not been extracted by NaHCO 3. 4. 4. It is suggested that these differences are connected with structural differences in the Z-line of cross-striated muscles of scallop and rabbit.

Reversible Dissociation of Ribonucleic Acid

THE work reported here was prompted by the observation that when infectious ribonucleic acid, isolated from Ehrlich ascites cells infected with Mengo encephalomyelitis virus1, was incubated with crystalline bovine plasma albumin in solutions of physiological ionic strength, its biological activity was lost or sharply inhibited. No loss of infectivity was observed when mixtures of these two substances were incubated in concentrated solutions of sodium chloride (0.6–1.0 M). However, infectivity was not regained when protein–ribonucleic acid mixtures in physiological saline were made 0.64 M with respect to sodium chloride. The biological activity of the Mengo ribonucleic acid and of the Mengo ribonucleic acid–bovine plasma albumin mixtures was assayed by a plaque method employing monolayers of Earle's L strain mouse fibroblasts2.