Conformational and phase transitions in lysozyme-thermosensitive polyelectrolyte complexes

The interaction of a small globular protein, lysozyme, with a thermosensitive N-isopropylacrylamide-sodium styrene sulfonate copolymer at pH 4.6 was studied by high-sensitivity differential scanning calorimetry. It was shown that, under these conditions, the copolymer and the protein are involved in formation of polyelectrolyte complexes. It was demonstrated that complexation affects the conformational state of lysozyme. One heat capacity peak attributed to protein denaturation or two well-resolved peaks related to denaturation of free and bound proteins were observed in the DSC curves depending on the mixture composition. For both forms of lysozyme, denaturation parameters (temperature and enthalpy) were determined as a function of mixture composition. For mixtures with low lysozyme contents, the above parameters of bound protein denaturation were independent of the mixture composition. At higher protein contents, these parameters increased with a rise in the protein content. The binding isotherm for the protein with the copolymer was obtained from calorimetric data at 64 ± 1°C. An analysis of the isotherm suggests that the native protein is bound to 24 equivalent binding sites of the polymer matrix. It was established that there are nearly 10 charged units of the copolymer per protein molecule in the native conformation. Lysozyme in the unfolded conformation additionally interacts with hydrophobic groups of the copolymer.     

Electrostatics of PEGylated micelles and liposomes containing charged and neutral lipopolymers

The electrostatics of large unilamellar vesicles (LUVs) of various lipid compositions were determined and correlated with steric stabilization. The compositional variables studied include (a) degree of saturation, comparing the unsaturated egg phosphatidylcholine (EPC) and the fully hydrogenated soy phosphatidylcholine (HSPC) as liposome-forming lipids; (b) the effect of 40 mol % cholesterol; (c) the effect of mole % of three methyl poly(ethylene glycol) (mPEG)-lipids (the negatively charged mPEG-distearoyl phosphoethanolamine (DSPE) and two uncharged lipopolymers, mPEG-distearoyl glycerol (DSG) and mPEG-oxycarbonyl-3-amino-1,2-propanediol distearoyl ester (DS)); and (d) the negatively charged phosphatidyl glycerol (PG). The lipid phases were as follows: liquid disordered (LD) for the EPC-containing LUV, solid ordered (SO) for the HSPC-containing LUV, and liquid ordered (LO) for either of those LUV with the addition of 40 mol % cholesterol. The LUV zeta potential and electrical surface potential (psi(0)) were determined. It was found that progressive addition of mPEG(2k)-DSPE to liposomes increases negative surface potential and reduces surface pH to a similar extent as the addition of PG. However, due to the "hidden charge effect", zeta potential was more negative for liposomes containing PG than for those containing mPEG(2k)-DSPE. Replacing mPEG-DSPE with mPEG(2k)-DS or mPEG-DSG had no effect on surface pH and surface potential, and zeta potential was approximately zero. Addition of low concentrations of cationic peptides (protamine sulfate and melittin) to PG- or mPEG-DSPE-containing liposomes neutralized the liposome negative surface potential to a similar extent. However, only in liposomes containing PG, did liposome aggregation occur. Replacing the negatively charged lipopolymer mPEG-DSPE with the neutral lipopolymers mPEG-DS or mPEG-DSG eliminates or reduces such interactions. The relevance of this effect to the liposome performance in vivo is discussed.     

Aggregation and fusion of phospholipid vesicles

Membrane fusion and aggregation of phospholipid vesicles are reviewed and discussed. The fusion process is viewed as consisting of several stages: aggregation and close apposition of the particles, destabilization, and finally, merging of the bilayers. A procedure is presented which yields both the rate constant of the dimerization (C₁₁) and the rate constant for fusion of the dimers (f₁₁), which is a direct measure of the probability that two apposed vesicles will fuse. Experimental methods used in the study of membrane fusion are reviewed, primarily with respect to their capacity to monitor the kinetics of vesicle fusion. A few kinetic studies on the mixing of aqueous contents, leakage and increase in size of fusing vesicles are presented in detail.The range of C₁₁ values for Ca²⁺-induced aggregation and fusion of small unilamellar vesicles (SUV, ～ 125 Å radius) composed of phosphatidylserine (PS) is 10⁶ to 5 × 10⁷ M^-1 in the presence of Ca²⁺ concentrations from 1.15 to 2 mM, respectively. For larger PS vesicles (LUV, ～ 500 Å radius) C₁₁ = 6.5 × 10⁷ M^-1s^-1 in the presence of 5 mM Ca²⁺. These values are in good agreement with theoretical calculations based on van der Waals and electrostatic interactions, in which binding of cations is explicitly taken into account. The rate constants of fusion, f₁₁, are 5 s^-1 for PS SUV and 0.08 s^-1 for LUV in the presence of 2 mM and 5 mM Ca²⁺, respectively. The significance of these fusion rate constants to the duration of the fusion event is discussed.Factors affecting fusion such as cations, temperature, membrane composition vesicle concentration and size are reviewed and analyzed. Di- or tri-valent cations induce fusion of acidic phospholipid vesicles (except for phosphatidylinositol) in either pure or mixed form. Among the neutral phospholipids, phosphatidylcholine (PC) inhibits but phosphatidylethanolamine (PE) sustains or enhances the fusion capacity of acidic phospholipid vesicles. Monovalent cations induce reversible aggregation of negatively charged vesicles, but they inhibit the fusion induced by divalent cations such as Ca²⁺ or Mg²⁺. Fusion of neutral phospholipid vesicles, and it occurs the cation-induced fusion of acidic phospholipid vesicles, and it occurs only at temperatures below the gel to liquid crystalline phase transition temperature Tc. This is in contrast to the acidic phospholipid vesicle fusion which is greatly enhanced when the temperature is above the Tc of the phospholipid.     

Influence of microenvironment and liposomal formulation on secondary structure and bilayer interaction of lysozyme

The conformation of peptide and protein drugs in various microenvironments and the interaction with drug carriers such as liposomes are of considerable interest. In this study the influence of microenvironments such as pH, salt concentration, and surface charge on the secondary structure of a model protein, lysozyme, either in solution or entrapped in liposomes with various molar ratios of phosphatidylcholine (PC):cholesterol (Chol) was investigated. It was found that entrapment efficiency was more pronounced in negatively charged liposomes than in non-charged liposomes, which was independent of Chol content and pH of hydration medium. The occurrence of aggregation, decrease in zeta potential, and alteration of ³¹P NMR chemical shift of negatively charged lysozyme liposomes compared to blank liposomes suggested that the electrostatic interaction plays a major role in protein–lipid binding. Addition of sodium chloride could impair the neutralizing ability of positively charged lysozyme on negatively charged membrane via chloride counterion binding. Neither lysozyme in various buffer solutions with sodium chloride nor that entrapped in liposomes showed any significant change in their secondary structures. However, significant decrease in α-helical content of lysozyme in non-charged liposomes at higher pH and salt concentrations was discovered.     

Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH

A sugar-based (reduced glucose) gemini surfactant forms vesicles in dilute aqueous solution near neutral pH. At lower pH, there is a vesicle-to-micelle transition within a narrow pH region (pH 6.0-5.6). The vesicles are transformed into large cylindrical micelles that in turn are transformed into small globular micelles at even lower pH. In the vesicular pH region, the vesicles are positively charged at pH < 7 and exhibit a good colloidal stability. However, close to pH 7, the vesicles become unstable and rapidly flocculate and eventually sediment out from the solution. We find that the flocculation correlates with low vesicle zeta-potentials and the behavior is thus well predicted by the classical DLVO theory of colloidal stability. Surprisingly, we find that the vesicles are easily redispersed by increasing the pH to above pH 7.5. We show that this is due to a vesicle surface charge reversal resulting in negatively charged vesicles at pH > 7.1. Adsorption, or binding, of hydroxide ions to the vesicular surface is likely the cause for the charge reversal, and a hydroxide ion binding constant is calculated using a Poisson-Boltzmann model.     

Solution Structure and Model Membrane Interactions of P‐113, a Clinically Active Antimicrobial Peptide Derived from Human Saliva

P‐113, AKRHHGYKRKFH‐NH₂, was derived from human saliva and found to possess clinical activity against fungus infections in HIV patients with oral candidiasis. We have determined the solution structure of P‐113 bound to membrane‐mimetic SDS micelles by two‐dimensional NMR methods. The SDS micelle‐bound structure of P‐113 adopts an α‐helical segment and the positively charged residues are clustered together to form a hydrophilic patch. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy and microcalorimetry, were used to show that P‐113 interacted with negatively charged phospholipid vesicles and induced dye release from these vesicles. However, its dye leakage efficiency is much less than the results of previously reported antimicrobial peptides. These results suggest that the antimicrobial activity of P‐113, unlike other antimicrobial peptides, may act not only through binding to and destabilization of the microbial membrane but also through a specific protein receptor on the microbial cell surface.     

Alkylated chitosans of low molecular weight as non-viral transfection vectors for gene therapy

Low molecular weight chitosans (5 kDa) hydrophobically modified with 3, 10, and 18 mol % of tetradecenoyl (TDC) groups have been synthesized. Their good solubility at neutral pH, their surface activity and micelle-forming properties as well as their ability to interact with negatively charged phospholipid vesicles mimicking the internal layer of cell plasma membranes, allow us to consider them as potential non-viral transfection vectors for gene therapy.     

Monitoring Local pH of Membranous Aggregates via Ratiometric Color Changing Response

A series of oxidized di(indolyl)arylmethanes (DIAM) with polyaromatic signaling moieties have been designed for monitoring local pH at the interfacial region of surfactant aggregates, such as micelles and vesicles. The oxidized DIAMs show changes in solution color from red to yellow when incorporated in cationic surfactants (at pH 7.4) and yellow to reddish pink when exposed to negatively-charged surfactants (at pH 5.0). The changes in surface charge can influence the interfacial pH (distinct from bulk pH of the medium) of the surfactant aggregates. The mechanistic studies indicate that the red-shifted absorption maxima observed in the presence of anionic amphiphiles (acidic local pH) originated from the protonated species. On the contrary, maxima in the blue region, triggered by positively charged amphiphiles (basic local pH), is attributed to the zwitterionic species. Such prototropic equilibrium affects charge transfer states of the molecules along with their self-assembly properties. Thus, it is evident that probes can predict as well as quantify the local pH change using the pseudophase ion exchange formalism. Also, the probes can detect the presence of anionic amphiphiles even when bound to phospholipid membranes.     

High capacity, charge-selective protein uptake by polyelectrolyte brushes

Surface plasmon resonance was used to measure binding of proteins from solution to poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes end-grafted from gold surfaces by atom transfer radical polymerization (ATRP). PDMAEMA brushes were prepared with a variety of grafting densities and degrees of polymerization. These brushes displayed charge selective protein uptake. The extent of uptake for net negatively charged bovine serum albumin (BSA) scaled linearly with the surface mass concentration of grafted PDMAEMA, regardless of grafting density. BSA was bound at a constant ratio of 120 DMAEMA monomer units per protein molecule for all brushes examined. The equivalent three-dimensional concentration of BSA bound in the brush (i.e., the bound BSA surface excess concentration divided by the brush thickness) decreased monotonically with decreasing grafting density. The concentration of BSA bound within brushes prepared at higher grafting densities was comparable with the aqueous protein solubility limit. BSA desorption from the brush required changes in solution pH and/or ionic strength to eliminate its net electrostatic attraction to PDMAEMA. Net positively charged lysozyme was completely rejected by the PDMAEMA brushes.     

Membrane curvature effects on glycolipid transfer protein activity

The glycolipid transfer protein (GLTP) is monomeric in aqueous solutions, and it binds weakly to membrane interfaces with or without glycolipids. GLTP is a surface-active protein and adsorbs to exert a maximal surface pressure value of 19 mN/m. The change in surface pressure following GLTP adsorption decreased linearly with initial surface pressure. The exclusion pressure for different phospholipids and sphingolipids was between 23 and 31 mN/m, being clearly highest for the negatively charged dipalmitoyl-phosphatidylserine. This can be explained by electrostatic forces when GLTP is positively charged at neutral pH (isoelectric point = 9.0) and by phosphatidylserine being negatively charged. If GLTP is injected under a palmitoyl-galactosylceramide monolayer above 30 mN/m, the presence of GLTP leads to a decrease in the surface pressure as a function of time. This suggests that GLTP is able to remove glycolipids from the monolayer without penetrating the monolayer. On the other hand, if phospholipid vesicles with or without glycolipids are also present in the subphase, no change in the surface pressure takes place. This suggests that GLTP in the presence of curved membranes is not able to transfer from or to planar membranes. We also show that transfer of fluorescently labeled galactosylceramide is faster from small highly curved palmitoyl-oleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine bilayer vesicles but not from palmitoyl-sphingomyelin vesicles regardless of the size.     

Transformative Supramolecular Vesicles Based on Acid-Degradable Acyclic Cucurbit[n]uril and a Prodrug for Promoted Tumoral-Cell Uptake

Smart supramolecular vesicles constructed by host–guest interactions between “acid-degradable” acyclic cucurbit[n]uril (CB[n]) and a doxorubicin prodrug are reported. “Acid-degradable” acyclic CB[n] is a high-affinity host for several common antitumor drugs, and its degradation leads to a more dramatic decrease in binding affinity than that observed for “acid-sensitive” hosts. Supramolecular complexation between acid-degradable acyclic CB[n] and a doxorubicin prodrug resulted in the formation of negatively charged supramolecular vesicles. The prodrug strategy allowed doxorubicin to be conjugated to vesicles in a stable manner with a high drug-loading ratio of 20 %. The resulting supramolecular vesicles were responsive to tumor acidity (pH 6.5). Induced by mildly acidic conditions (pH 6.5–5.5), acid-degradable acyclic CB[n] could be degraded, and this led to a vesicle-to-micelle transition to form positively charged micelles. This transition resulted in a pH-dependent change in size and surface charge, which improved tumoral-cell uptake for doxorubicin.     

An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.     

High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of X(PA) = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.     

Stability of phospholipid vesicles studied by asymmetrical flow field-flow fractionation and capillary electrophoresis

The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20 mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100 nm) prepared by extrusion at 25 °C were used. Phospholipid vesicles (liposomes) were stored at +4 and −18 °C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101 ± 3 nm and 122 ± 5 nm, respectively. No significant change in diameter was observed after storage at +4 °C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215 nm at peak maximum and mean diameters, respectively). When EPC was stored at −18 °C, large particles with diameters of 700–800 nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20 mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250–350 nm appeared during storage at +4 and −18 °C for more than 1 day.

The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and −18 °C, were studied at 25 °C. The performances of the coatings were further evaluated with three uncharged compounds. Only minor differences were observed between the same phospholipid coatings, showing that phospholipid coatings in CE are relatively insensitive to storage at +25, +4 °C or −18 °C.     

The adsorption of polyampholytes on negatively and positively charged polystyrene latex

The adsorption of two polyampholytes (a random copolymer of -glutamic acid and -lysine, and a well-defined tetramer of -lysyl- -glutamyl-glycine) onto positively and negatively charged latex was studied as a function of the pH and the ionic strength. The adsorbed amount proved to be almost independent of the salt concentration. The pH dependence was found to follow the same trends on negatively charged and positively charged latex. At low pH, where the polyampholytes are positively charged, a high adsorbed amount was found irrespective of the sign of the surface charge. At high pH, where the macromolecules are negatively charged, no adsorption was measured, not even with the positive latex. This is probably due to the very good solubility of the polyampholytes at this pH. Electrophoretic mobility measurements revealed that already at very low concentrations of polyampholyte charge reversal of the particles occurred.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID VESICLE SYSTEMS: COMPARISON OF INSIDE-OUTSIDE ASYMMETRY BETWEEN LARGE AND SMALL UNILAMELLAR VESICLES*

Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV.     

Thermodynamic study of small hydrophobic ions at the water-lipid interface

The thermodynamics of binding of two small hydrophobic ions such as norharman and tryptophan to neutral and negatively charged small unilamellar vesicles was investigated at pH 7.4 using fluorescence spectroscopy. Vesicles were formed at room temperature from dimyristoyl phosphatidylcholine (DMPC) or DMPC/dimyristoylphosphatidic acid and DMPC/dimyristoylphosphatidylglycerol. The changes in fluorescence properties were used to obtain association isotherms at variable membrane surface negative charge and at different ionic strengths. The binding of both ions was found to be quantitatively enhanced as the percentage of negative phospholipid increases in the membrane. Also, a decrease in ion binding was found to occur as the concentration of monovalent salt was increased (0.045-0.345 M). If electrostatic effects were ignored, the experimental data showed biphasic behavior in Scatchard plots. When electrostatic effects were taken into account by means of the Gouy-Chapman theory, the same data yielded linear Scatchard plots that were described by a simple partition equilibrium of the hydrophobic ion into the lipid-water interface. We demonstrate that the effective interfacial charge, nu, of the ion is a determinant factor to obtain a unique value of the intrinsic (hydrophobic) binding constant independently of the surface charge density of the lipid membrane.     

Adsorption of a cationic surfactant, miramistin, from aqueous solutions on the surface of highly dispersed silica

The adsorption of a universal antiseptic agent, the cationic surfactant miramistin, on the surface of highly dispersed silica has been studied. It has been shown that, when miramistin is adsorbed from acidic premicellar solutions, the main contribution to miramistin binding with the surface is made by hydrogen bonding between amide groups of surfactant molecules and silanol groups of silica, which is, at higher pH values, accompanied by ionic interaction between positively charged quaternary nitrogen atoms of miramistin and negatively charged dissociated silanol groups. In the case of adsorption from a micellar solution, an increase in the surface concentration of miramistin is almost independent of solution pH, because the second layer is predominantly formed due to hydrophobic interactions.     

Oxide-dependent adsorption of a model membrane phospholipid, dipalmitoylphosphatidylcholine: bulk adsorption isotherms

The importance of substrate chemistry and structure on supported phospholipid bilayer design and functionality is only recently being recognized. Our goal is to investigate systematically the substrate-dependence of phospholipid adsorption with an emphasis on oxide surface chemistry and to determine the dominant controlling forces. We obtained bulk adsorption isotherms at 55 degrees C for dipalmitoylphosphatidylcholine (DPPC) at pH values of 5.0, 7.2, and 9.0 and at two ionic strengths with and without Ca(2+), on quartz (alpha-SiO(2)), rutile (alpha-TiO(2)), and corundum (alpha-Al(2)O(3)), which represent a wide a range of points of zero charge (PZC). Adsorption was strongly oxide- and pH-dependent. At pH 5.0, adsorption increased as quartz < rutile approximately corundum, while at pH 7.2 and 9.0, the trend was quartz approximately rutile < corundum. Adsorption decreased with increasing pH (increasing negative surface charge), although adsorption occurred even at pH > or = PZC of the oxides. These trends indicate that adsorption is controlled by attractive van der Waals forces and further modified by electrostatic interactions of oxide surface sites with the negatively charged phosphate ester (-R(PO(4)-)R'-) portion of the DPPC headgroup. Also, the maximum observed adsorption on negatively charged oxide surfaces corresponded to roughly two bilayers, whereas significantly higher adsorption of up to four bilayers occurred on positively charged surfaces. Calcium ions promote adsorption beyond a second bilayer, regardless of the sign of oxide surface charge. We develop a conceptual model for the structure of the electric double layer to explain these observations.     

Lysozyme adsorption studies at the silica/water interface using dual polarization interferometry

Lysozyme adsorption at the silica/water interface has been studied using a new analytical technique called dual polarization interferometry. This laboratory-based technique allows the build up or removal of molecular layers adsorbing or reacting on a lightly doped silicon dioxide (silica) surface to be measured in terms of thickness and refractive index changes with time. Lysozyme adsorption was studied at a range of concentrations from 0.03 to 4.0 g dm(-3) and at both pH 4 and pH 7. Adsorbed layers ranging from 14 to 43 +/- 1 A in thickness and 0.21 to 2.36 +/- 0.05 mg m(-2) in mass coverage were observed at pH 4 with increasing lysozyme concentration, indicating a strong deformation of the monolayer over the low concentration range and the formation of an almost complete sideways-on bilayer toward the high concentration of 4 g dm(-3). At pH 7, the thickness of adsorbed layers varied from 16 to 54 +/- 1 A with significantly higher surface coverage (0.74 to 3.29 +/- 0.05 mg m(-2)), again indicating structural deformation during the initial monolayer formation, followed by a gradual transition to bilayer adsorption over the high concentration end. The pH recycling performed at a fixed lysozyme concentration of 1.0 g dm(-3) indicated a broadly reversible adsorption regardless of whether the pH was cycled from pH 7 to pH 4 and back again or vice versa. These observations are in good agreement with earlier studies undertaken using neutron reflection although the fine details of molecular orientations in the layers differ subtly.