Circular dichroism of bovine serum albumin in divalent salt solutions

The circular dichroism of bovine serum albumin in divalent salt solutions was investigated. The salts selected were magnesium chloride and calcium chloride. Their effects on the secondary and tertiary structures of the protein were compared with that of lithium chloride. It is well known that the elements Ca, Mg, and Li have many properties in common. The results show the similarity of their ions in the capacity of deforming protein, in spite of their characteristic pharmacological functions.     

Mono- and divalent competitive adsorption to a charged membrane in a closed system: A comparative study

Competitive adsorption to a negative surface between monovalent and divalent cations is studied in a closed system. A self-consistent theory is presented for the cases when the divalent cation binds to two negative sites (1:2 binding) and to one negative site (1:1 binding). It is demonstrated that these two cases are distinguishable when the relative difference in total divalent concentrations determined at fixed bulk divalent concentrations is plotted as a function of the bulk monovalent concentration. The 1:2 binding case yields a sigmoidal curve while the 1:1 binding curve is hyperbolic. The comparison between the 1:2 and 1:1 binding cases of the divalent cation is extended to include; (1) the existence of a surface charge to which no binding occurs, and (2) the case when an additional non-binding monovalent cation is present.     

Perspectives to produce positively or negatively charged polyhydroxyalkanoic acids

An overview is provided on the possibilities of producing positively and negatively charged poly(β-hydroxyalkanoates), PHAs. A large variety of bacterial polyesters with functionalized terminal side chains can be produced in microbial fermentation processes by a direct polymerization of respective carbon sources, that is, carbon sources that carry functional groups in their ω-position. However, charged PHAs are not accessible by a direct approach and must be synthesized via polymer-analogous reactions of functionalized bacterial polyesters. PHA polyanions are produced by converting the terminal functional groups into carboxylate groups, while PHA polycations are produced by introducing terminal amino groups. PHAs with terminal vinyl groups emerged as most suitable PHA precursors, as they can be produced in relatively high yields and the double bonds are sufficiently reactive. The oxidation of vinyl groups yields PHA polyanions. The conversion of terminal vinyl groups into epoxides with a subsequent ring-opening reaction with an amine yields PHA polycations. Other functionalized PHA that potentially lend themselves to polymer-analogous reactions are reviewed.     

Size and shape of charged micelles of ionic surfactants in aqueous salt solutions

Light-scattering has been measured on aqueous NaCl solutions of dodecyldimethylammonium chloride and sodium dodecyl sulfate. From molecular weight determination it is confirmed that spherical micelles are formed at low NaCl concentrations, but at high NaCl concentrations the small micelles formed at the critical micelle concentration further associate to form large rod-like micelles with increasing micelle concentration. The reduction of repulsion between charged groups induces the sphere-rod transition of micelle shape. The dependence of molecular weight on ionic strength can be expressed by double logarithmic relations, which are dependent on the micelle shape. While dodecyldimethylammonium chloride dissolves even in 4.00 M NaCl, sodium dodecyl sulfate solutions exhibit some XXX in angular dissymmetry at NaCl concentrations higher than 0.50 M at low temperatures.     

Dimethonium,a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes

Calcium and other alkaline earth cations change the electrostatic potential adjacent to negatively charged bilayer membranes both by accumulating in the aqueous diffuse double layer adjacent to the membrane and by adsorbing to the phospholipids. The effects of these cations on the electrostatic potential are described adequately by the Gouy-Chapman-Stern theory. We report the results of experiments with ethane-bis-trimethylammonium, a cation that has been termed "dimethonium" or "ethamethonium" in analogy with hexamethonium (hexane-1,6-bis-trimethylammonium) and decamethonium (decane-1,10-bis-trimethylammonium). We examined the effect of dimethonium on the zeta potential of multilamellar vesicles formed from the negative lipid phosphatidylserine (PS) and from 5:1 phosphatidylcholine/phosphatidylserine mixtures in solutions containing 0.1, 0.01 and 0.001 M sodium, cesium, or tetramethylammonium chloride. We also examined the effect of dimethonium on the conductance of planar PS bilayer membranes and the 31P NMR signal from sonicated PS vesicles formed in 0.1 M NaCl. We found no evidence that dimethonium adsorbs specifically to bilayer membranes. All the results, except for those obtained with vesicles of low charge density formed in a solution with a high salt concentration, are consistent with the predictions of the Gouy-Chapman theory. We conclude that dimethonium, which does not have the pharmacological effects of hexamethonium and decamethonium, is a useful divalent cation for physiologists interested in investigating electrostatic potentials adjacent to biological membranes.     

Covalent binding of negatively charged phospholipid to Na,K-ATPase

Phosphatidylglycolaldehyde and its lyso derivative were applied as probes in order to study lipid-protein interactions with purified, membrane-bound Na,K-ATPase. Reduction with [3H]NaCNBH3 led to formation of a stable chemical derivative between added lipid and protein. The extent of modification of the two subunits of Na,K-ATPase was similar. Extensive tryptic digestion of derivatized ATPase resulted in cleavage of the alpha-subunit without hydrolysis of the beta-subunit.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces

The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent < divalent < trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups.We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding.The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.     

The adsorption of divalent cations to phosphatidylcholine bilayer membranes

Electrophoretic mobility and ³¹P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations to phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the ³¹P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the ³¹P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Sr²⁺, Ba²⁺. If we assume that a lipid molecule occupies an area of 60 Ų and that there is a 1: 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.     

The adsorption of divalent cations to phosphatidylglycerol bilayer membranes

The ability of the Stern equation to describe the adsorption of divalent cations to phosphatidylglycerol membranes was tested by combining ³¹P-NMR and electrophoretic mobility measurements. In 0.1 M sodium chloride both the ³¹P-NMR and the zeta potential data are well described by the Stern equation. ³¹P-NMR and ¹³C-NMR results indicate that cobalt forms inner-sphere complexes only with the phosphate group of phosphatidylglycerol molecules and that a substantial fraction of the adsorbed cobalt ions form outer-sphere complexes. Evidence is presented that suggests the alkaline earth cations also bind to phospholipids mainly by forming outer sphere complexes. Electrophoretic mobility measurements were performed with several different divalent cations. In all cases the zeta potentials in 0.1 M sodium chloride were well described by the Stern equation. The intrinsic 1 : 1 association constants (M^?1) for the phosphatidylglycerol complexes decreased in the sequence: Mn²⁺, 11.5; Ca²⁺, 8.5; Ni²⁺, 7.5; Co²⁺, 6.5; Mg²⁺, 6.0; Ba²⁺, 5.5 and Sr²⁺, 5.0.     

Protein adsorption to solid surfaces.

The phenomenon of protein adsorption to solid surfaces affects the performance of many materials and processes, in areas ranging from medicine to biochemical engineering. Controlling protein adsorption, from solutions of single proteins as well as from more complex mixtures, requires an understanding of the mechanism(s) by which it occurs. This, in turn, entails detailed characterization of both the protein and the solid surface and identification of those factors controlling the adsorption process.     

Bacterial adsorption to thermoresponsive polymer surfaces

The ability of microorganisms to `recognise' a change in the hydrophobicity/hydrophilicity balance of the surface was demonstrated using thermoresponsive poly(N-isopropylacrylamide) co-polymers with different Lower Critical Solution Temperatures. The polymers were grafted onto hydrolysed glass under well controlled conditions and the adhesion was followed using ¹³C-labelled Listeria monocytogenes. Attachment of the bacteria was found to be directly affected by the polymer transition from a hydrophilic to a hydrophobic state but by less than one order of magnitude.     

Thioridazine induces erythrocyte stomatocytosis due to interactions with negatively charged lipids

Despite the fact that thioridazine is used clinically as a neuroleptic drug, little is known about the molecular mechanisms underlying its biological effects, in particular about its interactions with membranes. In the present work we investigate the influence of thioridazine on model and cell membranes, using calorimetry, DPH fluorescence polarization measurements, studies of haemolysis and scanning electron microscopy. The experiments show that thioridazine interacts with lipid bilayers and intercalates into bilayer structure. We found that erythrocyte stomatocytosis induced by the drug might be related to preferential interaction of thioridazine with charged lipids.     

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces.

The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Sequential adsorption of F(ab')(2) and BSA on negatively and positively charged polystyrene latexes

The aim of the present work is to study the sequential adsorption of F(ab')(2) and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab')(2) on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab')(2) molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab')(2) and monomeric BSA were performed at two different pre-adsorbed F(ab')(2) amounts on both types of latex. Displacement of F(ab')(2) occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab')(2) previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab')(2) on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. (c) 1995 John Wiley & Sons, Inc.     

A mechanism of translocation based on high proton mobility and K+ counterflux at negatively charged surfaces in sieve elements

Large pH gradients between the sources and the sinks involved in translocation of metabolites arise owing to photosynthesis and nitrate reduction in the leaves and respiration in the sinks. pH equalization between the sinks and the sources is proposed to be brought about by a rapid movement of H⁺ from sinks to sources along the negatively charged surfaces lining the translocation pathways and the ray symplast. This movement is made possible by a charge-compensating movement of K⁺ in the electrical double layer. In the sieve tubes and specially at the sieve plate pores the movement of K⁺ in the diffused layer is suggested as the driving force for translocation of metabolites. In the transport network of plants K⁺ moves in loops, acting like a conveyor belt in the phloem. The proposed mechanism explains all experimental observations related to translocation and also solves the problem of pH-stating in the source and sink cells. Its implications for shoot-root cooperation have been also indicated.