Elastic compliance and adsorption profiles of Bovine serum albumin at fluid/solid interface in the presence of electrolytes.

Non-trivial topology of proteins under shear suggests that even small structural changes in proteins result in dramatic variations in the mechanical properties and stability. In this study, we have analysed the elastic compliance of solvated bovine serum albumin (BSA) with NaCl,MgCl2, FeCl3 of concentration-ranging from 50 mM to 250 mM using Quartz crystal microbalance with dissipation. The compliance shows a reverse Hofmeister trend (Na + 

Plasticity or elasticity? Relating elastic moduli with secondary structural features of mixed films of polypeptides at air/fluid and fluid/solid interfaces.

Viscoelastic properties of molecular assemblies formed by mixing poly (l-lysine) (PLL) and poly (l-glutamic acid) (PGA) at pH = 7.5 have been studied using Quartz crystal microbalance with dissipation (QCM-D). The inter-molecular complex between PLL and PGA arising from strong electrostatic interaction, leads to local changes in secondary structure resulting in intra-molecular complexes. ATR-FTIR analysis of the Secondary structural features of PLL, suggest an abundance of anti-parallel beta sheet that causes a significant change in the morphology of the self-assembled structures. A combination of spectroscopy, Brewster angle microscopy (BAM), Transmission electron and scanning electron microscopy show that an inverse relationship exists between the elasticity of the different PLL + PGA mixed films and the % anti parallel beta sheet conformation. The elastic moduli for the mixtures change from about 0.913 ± 0.01 GPa for pure PLL to about 0.764 ± 0.01 GPa in the mixture when PGA increases. The localized breaking and reformation of the ion pairs in the complex control their sizes and show an inverse relationship with the elastic moduli. The rheological profiles of the films, elastic moduli, together with their surface morphology from microscopy (TEM) and (SEM) confirmed their increasing propensity to self-assemble in one dimension to form tapes, colloidal particles and their composite assemblies.

Sub-micron sized cytochrome c particles adsorbing to solid surfaces: A comparison between solution phase and colloidal system.

Present work reports on Cytochrome C (Cyt C) adsorption from solution and as sub-micron sized colloidal particles (pH 7.5) at fluid/solid interface. The colloidal particles from 2 methods ((method 1 freeze-thaw); (method 2-Decane/water interface)) have been characterized by UV-Visible spectroscopy, Static and Time resolved Fluorescence spectra and Circular dichroic spectroscopy. Morphology and size studies indicate that the colloids are solid and well-packed. Using Quartz crystal microbalance with dissipation, elastic compliance of the protein on quartz surface has been monitored. Properties of the protein from solution and as colloids assembled from method 2 are similar in elastic compliance (65.53 ±â€¯1.2 and 73.5 ±â€¯1.1 GPa-1 respectively) due to polar/non-polar interactions at the solid surface. For particles from method 1, irregular desolvation of water on the particle surface results in higher compliance (104.3 ±â€¯1.3 GPa-1). Change in work of adhesion from contact angle profiles shows optimal adhesion for colloids from method 1 whereas the protein solution and as colloids from method 2, show subnormal adhesion. The work shows the role of extensive H-bonding with the hydrodynamically coupled water leading to a fluid like system in protein colloids from method 1 whereas those from method 2 behaved more like the pure protein adsorbing from solution.

Elastic compliance as a tool to understand Hofmeister ion specific effect in DMPC liposomes.

Elastic compliance of DMPC liposomes with Hofmeister electrolytes: NaCl, Na2SO4, Na2CO3, NaNO3, KCl and MgCl2 studied using Quartz crystal microbalance with dissipation has been correlated with changes in their lamellar spacing from SAXS. The study suggests that hydration water of the different ions has an effect on the overall packing of the lipid bilayer that results as either a dehydrated liposome or where water smears the surface of the liposomes. Ratio of hydrogen bonded carbonyl and phosphate of polar region of the liposomes from ATR-FTIR spectroscopy, suggests that the polar groups are less hydrated due to the displacement of water by the electrolytes compared to pure DMPC and ordered in the sequence for cations as: K+ < Na+,Mg2+ and for anions as SO42- < CO32- < Cl- < NO3-. These findings show the usefulness of Elastic compliance for structural studies of composite phospholipid bilayers, lipid-protein complexes and lipid systems of reduced dimensionalities.

Colloids versus solution state adsorption of proteins: Interaction of Myoglobin with supported lipid bilayers.

This study examines adsorption of Myoglobin (Mb) in solution and as colloid on supported lipid bilayers of neutral phospholipids and a mixture of neutral+cationic lipids formed on gold coated quartz in a Quartz crystal microbalance (QCM). Results indicate that thin adsorbed films of Mb in solution and as colloids, show atleast 3 steps in the interaction with the bilayers: i) An initial strain of a viscoelastic film ii) Entrained water that moves in and out of the adsorbed film and iii) The coupled load from the bulk liquid which increases the strain of the film. These three components constitute an effective viscoelastic film which is rigidly coupled to the QCM. Grazing incidence XRD (GIXD) shows that the bilayer head group remains nearly undisturbed for Mb solution with pure (neutral) and (neutral+cationic) mixtures, whereas for the colloids there is an increase in head group thickness with neutral and decrease in the case of mixture. Unsaturation in the alkyl tails in the neutral lipid resulting in flexible disordered bilayers and more entrained water in the cationic system results in these changes. The sensitivity of QCM-D, makes it useful to study real-time monitoring of bilayer structural robustness cytotoxicity, drug delivery and lipid self-assembly.

Protein microcapsules: preparation and applications.

Liposomes and polymerosomes generally represent the two most widely used carriers for encapsulating compounds, in particular drugs for delivery. While these are well established carriers, recent applications in biomedicine and food industry have necessitated the use of proteins as robust carriers that are stable under extreme acidic and basic conditions, have practically no toxicity and are able to withstand high shear force. This review highlights the different methods for using proteins as encapsulating materials and lists some biomedical applications of the microcapsules. The advantages and limitations in the capsules from the different preparation routes are enumerated.

Microviscosity-induced conformational transition in ß-lactoglobulin in the presence of an ionic liquid.

This study reports on the helix-beta conformation transition of bovine ß-lactoglobulin (ßLG) prepared at two different pH conditions (pH 4 and 7.5) and in the presence of the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate (IL-emes). The investigation was carried out by combining a range of techniques such as circular dichroic (CD) spectroscopy, steady-state fluorescence spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy. The influence of microviscosity induced by IL-emes on the secondary structure of ßLG was studied using a quartz crystal microbalance and correlated with the steady-state fluorescence emission. The effect of heat on the helix-beta transition in ßLG was directly measured by ITC by titrating ßLG with IL-emes. The net effect of heat after subtraction of the heat of dilution was negative in both cases, suggesting that the protein moves to a stable conformation. The changes in the overall aggregated structures were confirmed by transmission electron microscopy, where a shift in the size and morphology of aggregates was found, from large clusters (size of 70 nm) at pH 4 to smaller aggregates (size of 20 nm) at pH 7.5, which reduced to 7 nm in the presence of the IL. The transformation of helical to beta structure at pH 4 show that the folding pathway in the presence of the ionic liquid is hierarchical, whereas at neutral pH, it appeared to be nonhierarchical and the final native structure was acquired by nonlocal interactions through typical forces involved in the stabilization of the tertiary structure.

Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete ß sheet from its native helical conformation. Rehydration or dilution reverses the ß sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 µM, the protein changes from ß to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure-molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir-Blodgett film technique show layered morphology. This study shows that the conformation of Mb is completely reversible going from ß â†’ helix → ß sheet up to 200 µM of Phe-IL. Similar surface tension values for Mb in water and in Phe-IL suggests that direct ion binding interactions with the protein coupled with the change in local viscosity from the IL seems to not only alter the secondary structure of individual proteins but also drives the self-assembly of the protein molecules leading finally to fibril formation.

Evaluation of hydropathy of amino acids from a comparison of their viscosities inside vesicles and on supported lipid bilayers.

The viscosity of amino acids enclosed in giant lipid vesicles (η(out)) subjected to a shear flow near a solid surface has been studied using quartz crystal microbalance (QCM). This viscosity has been compared with shear viscosity for the different amino acids adsorbed on supported bilayers (SLBs) (η(in)) of the lipids on quartz. Using a first approximation of vesicles as model rigid spheres, the measured viscosities and the extent of deformation of vesicles observed using optical microscopy, two non-dimensional parameters: the reduced volume and the ratio of (η(in))/(η(out)) have been analyzed as a function of physical parameters: vesicle substrate distance (vesicle vs. supported lipid bilayers), vesicle size and their variation as a function of the viscosity. The kinematics of the vesicles with the amino acids compared with the shear at supported lipid bilayers seems to describe a reasonable hydropathy scale for the amino acids. The results show that there is a direct correlation between the above parameters and the polarity variations in amino acids suggesting that the viscous force may be an important parameter and should be taken into account in studies on membrane proteins interacting with cells and cell adhesion in flow chambers where cell membrane and the adhesive substrate are in relative motion.

Real-time monitoring of invertase activity immobilized in nanoporous aluminum oxide.

In this work, we demonstrate the activity of enzyme invertase immobilized in the pores of nanoporous anodized 3 µm thick aluminum oxide (AA). The porous anodic alumina has uniform nanosized pores with an interpore distance of p = 100 nm, with pore diameters on the order of 60-65 nm. The pores trap the enzyme and continuous monitoring of the activity is carried out in a flow cell where the substrate is made to flow and the product is detected. The activity of the immobilized enzyme has been determined for the different concentrations of sucrose and for pH ranging from 3 to 6.5. Maximum activity was found for pH 4.5. Adsorption of the enzyme followed by its interaction with the substrate have been analyzed using confocal laser scanning microscopy (CLSM) and surface plasmon spectroscopy (SPR) and the results obtained show excellent correlation. SPR results show a biphasic kinetics for the adsorption of the enzyme as well as its interaction with the substrate with rates of adsorption for the enzyme at k = 2.9 × 10(5) M(-1) s(-1) and 1.17 × 10(5) M(-1) s(-1). The rate of interaction of the substrate with the invertase is initially rapid with k = 4.49 × 10(5) M(-1) s(-1) followed by a slower rate 1.43 × 10(4) M(-1) s(-1).

Influence of sequence on the self-assembly of peptide nanoribbons on silicon substrates.

This work reports the formation of stable nanoassemblies of short pentapeptides LKLKL (pepI) and their mutated sequence LKKLL (pepII) obtained from their Langmuir-Blodgett films transferred onto hydrophilic and hydrophobic silicon substrates. The adsorption and assembly of the LB films of these peptides on solid surfaces have been studied by quartz crystal microbalance, surface plasmon resonance, and scanning electron microscopy. Both pepI and pepII assemble into nanosized ribbons, with diameters around 20-25 nm and lengths greater than 5 µm on hydrophobic surface, and tend to aggregate on hydrophilic surfaces with pepII showing twisted structures. Circular dichroic spectra of the films on a hydrophobic surface showed formation of a ß-sheet-like structure, while the corresponding solution spectra did not show any specific secondary structure. Our results demonstrate the formation of a two-dimensional dense array of nanoassemblies with either vertical or horizontal patterns from such short peptides that may find application in nanotechnology.

Assembling fibrinogen at air/water and solid/liquid interfaces using Langmuir and Langmuir-Blodgett films.

Langmuir films of pure fibrinogen (Fg) and Fg spread at the air/buffer interface and subphase containing electrolytes, NaCl, KCl, CaCl(2), and ZnCl(2), have been analyzed to understand the role of the surface/interface in mediating the organization of the protein eventually to fibrils. These films have been characterized by the surface pressure and surface potential-molecular area ((pi-A) and (DeltaV-A)) isotherms and Brewster angle microscopy (BAM). The Langmuir-Blodgett (LB) films of the protein transferred to the solid substrates have been characterized by scanning electron microscopy (SEM) and circular dichroism (CD). Our results suggest that fibrils are formed during organization at air/solution interface and also in LB films. The rate of formation of the fibril is the maximum for Fg with ZnCl(2). Adsorption of Fg to surfaces coated with a neutral lipid, dimyristoylphosphatidylcholine (DMPC), and a cationic lipid, dioctadecyldimethylammonium bromide (DOMA), from a range of solution concentrations has been studied using a quartz crystal microbalance (QCM). The work of adhesion of the protein on the solid surface shows fibril formation and positive adhesion for Fg in the presence of electrolytes. SEM results show that the adherent protein exhibits the widely reported nodulelike structure in the presence of CaCl(2) and ZnCl(2). These results provide definite evidence that specifically designed surfaces can promote adhesion of Fg and also activate fibril formation even in the absence of thrombin.

Nanoparticles of nickel oxide and nickel hydroxide using lyophilisomes of fibrinogen as template.

Stable lyophilisomes of fibrinogen at pH 7.5 have been prepared by the method of a rapid freezing-heating and annealing sequence. Reduction of the lyophilisomes of the nickel-fibrinogen complex coated on solid substrates and subsequent heating showed formation of nickel hydroxide and finally nickel oxide. Ultraviolet-visible spectroscopy has been used to monitor the thin films of pure fibrinogen microcapsules, as well as the subsequent nucleation and growth of nanoparticles within the supramolecular structure. Transmission electron microscopy showed initially a thread-like structure which disappeared on continued heating, resulting in nanoparticles ranging from 10 to 50 nm. Particle-size distribution of product was analyzed by using X-ray diffraction (XRD), transmission electron microscopy (TEM) and the corresponding selected area electron diffraction (SAED), and Brunauer-Emmett-Teller (BET) N(2) adsorption. The results suggest that the NiO particles have a body-centered cubic structure and are well dispersed. The particle-size distribution ranges from 10 to 50 nm with an average particle size about 28 nm, and the specific surface area is 34 m(2)/g. Magnetic study carried out on the prepared nanoparticles showed a ferromagnetic behavior.

Structural influence of mono and polyhydric alcohols on the stabilization of collagen.

In the present study, solvents effects on the structure of collagen have been examined by circular dichroism and their interfacial tension at glass/liquid and Teflon/liquid. Changes in the conformations of the protein have been analyzed after equilibration with aqueous solutions of monohydric and polyhydric alcohols like methanol, ethanol, n-propanol, propane-2-diol and glycerol. The results from viscosity and Circular dichroism (CD) spectra suggest a clear distinction in the structural changes for collagen with monohydric alcohols as against polyhydric ones. The surface tension and interfacial tension at glass (high surface energy, HFSE) and Teflon (Low surface energy, LSFE) reflect similar differences between the monohydric and polyhydric alcohols. Studies on the interfacial energy of the adsorbed protein at glass/solution interface compared to that of Teflon/solution interface show that the water structure near glass gets perturbed leading to an increase in the average free energy of the bulk water phase and a reduction in hydrophobic effect near the glass. The results suggest that the different solvents alter the hydrophobic effect on the hydrated protein to different extent and thus influence folding equilibrium of the protein without directly interacting with it. Polyhydric alcohols seem to favor the native collagen structure while monohydric alcohols enhance it.

Solid-liquid interfacial energy as a tool to estimate shifts in isoelectric points of adsorbed proteins on solid surfaces.

This work reports the estimation of isoelectric points (pIs) of adsorbed amino acids and proteins on solid surfaces in the pH range between 3.5-11.0 from a measurement of solid/liquid interfacial energy. The values thus obtained are compared with the pIs determined in solution phase by other methods. Both glass and Teflon have been chosen as model solid surfaces. Close agreement between the reference pI values, obtained by the capillary isoelectric focusing and those obtained at solid/liquid interface is observed within an average difference of 0.04-0.08 pH unit when the pIs are above the pI of glass. For systems whose pIs are far away from that of glass (either in the acidic or highly alkaline range), a large shift in the isoelectric point is observed. In case of Teflon the pIs are closer to the reported values than at glass/liquid interface. This could be due to the fact that Teflon being a hydrophobic surface, its surface is dominated by dispersive forces, which may not be seriously affected by pH changes. The shift in the values at solid/liquid interface compared to that in solution have been examined using an 'image charge approach.'

Synthesis and surface properties of aqueous dispersions of poly(ester-imide) prepared from anhydride terminated polyester prepolymer and diisocyanate.

Aqueous dispersions of poly(ester-imide)s [P(E-I)s] have been prepared by dispersing the P(E-I)s in water without any external solubilizing agents. P(E-I)s were prepared from anhydride-terminated polyester prepolymer and diisocyanate. The -COOH groups in the polymer were then neutralized using triethylamine and the P(E-I)s were subsequently dispersed in water. The influence of the degree of ionization of polymers on the particle size and viscosity of the dispersion has been studied. The dispersions were crosslinked using polyaziridine. The crosslinked dispersion cast films were characterized for dynamic mechanical properties. As the ionic content increased the particle size decreased and the viscosity increased. When the amount of crosslinker added was varied, for a fixed percentage of ionization, the glass transition temperature Tg shifted to higher values. Critical surface tension (CST) measurements indicated reorganization of hydrophobic groups on the surface after crosslinking.

Oxidation of benzaldehyde in films by Oxo-Cr(V) salen derivatives at solid/liquid interface.

The oxidation of benzaldehyde as a film on a solid surface by various substituted oxochromium(V) salen in solution has been studied by monitoring the change in contact angle of the oxidant at the film/liquid interface utilizing a Teflon cell of known hydrodynamics and controlled convection/diffusion. The kinetics of the redox reaction in bulk has been monitored by measuring the change in absorbance of the oxidant solution. The interfacial study permits analysis of adsorption of the oxidant followed by the oxidation of the substrate under pseudo-first-order conditions. A comparison of the independent surface-averaged kinetic data with those obtained in the solution phase oxidation reaction is made and a model is presented for the mechanism of the interfacial reaction. The kinetic investigation shows that the rate of oxidation is accelerated in the presence of an electron-withdrawing group and is faster at the solid/liquid interface compared to the bulk. The probable mechanism of the redox reaction is discussed.

Influence of ammonium nitrate in phase transitions of Langmuir and Langmuir-Blodgett films at air/solution and solid/solution interfaces.

The effect of ammonium nitrate on the phase transitions in Langmuir films of amphiphiles-stearic acid, stearyl amine (STAM), stearyl alcohol, dihexadecylphosphate, and the quarternized ammonium salt dioctadecyldimethylammonium bromide have been studied at air/water interface and in local ordering of their Langmuir-Blodgett films (LB films). The study shows that except for the stearyl amine (STAM) all other monolayers exhibit a liquid-expanded to liquid-condensed transition with slight expansion in area in the presence of ammonium nitrate. STAM monolayers show a new phase transition, which possibly arises due to the differently ionized amino groups, and change in solvation sheath due to an ion-dipole type interaction between the amino groups and the ammonium ion in the subphase. Mixed films of the amine with the acid and alcohol did not show such intermediate phases indicating that competing H-bonds between polar groups themselves and dipolar couplings between the polar groups and ammonium nitrate play a major role in the organization of the molecules at the interface. The above effect resulting in a change in the local order is borne out by Brewster angle micrographs (BAM) of the Langmuir films of STAM at air/solution interface. Such behavior is also seen at solid/liquid interfaces where the polar component of surface energy undergoes a drastic change for the amine films transferred onto solid substrates from the air/ammonium nitrate solution interface.

Molecular dipoles at substrate/film interfaces influencing surface energy of Langmuir-Blodgett films.

Langmuir and Langmuir-Blodgett (LB) films of novel rigid Schiff base amphiphiles with different polar groups on aromatic rings have been studied at air/water interfaces and on solid substrates. The local surface potentials for the different substituents on the aromatic groups of the amphiphiles are correlated to the surface energy of LB films on solid substrates. Their contributions are probed on high-surface-energy (hsfe) quartz and hydrophobically modified low-surface-energy (lsfe) quartz. Using theoretical estimates, the long-range effects of the substituents of the polar groups on the alkyl chains have been studied from charge on the carbon and hydrogen atoms of the alkyl tails. The calculations indicate that a substituted carbon atom has a charge that is directly dependent on the nature of the atoms to which it is attached rather than on the charges of any other atoms of the substituents. This work shows that the polar substituents on the head group clearly influence the distal methylene groups of the tail. Thus, any treatment, either theoretical or experimental, of these films in contact with solid substrates should include the local relative permitivities of the polar moieties, which seem to play an important role in determining macroscopic properties such as surface energy.

Phase Transition in Langmuir Films of Octadecylmalonic Acid.

The phase transitions observed in the surface pressure-molecular area (pi-A) isotherms of monolayers of the dicarboxylic compound octadecylmalonic acid (OMA) at the air/water interface were investigated using molecular dynamics simulation in the molecular area ranging from 20 to 50 A2/molecule in a triangular lattice. Potential energy, torsion angles, and hydration of the head groups under periodic boundary conditions have been used for the study. Hydration was done by a stacked array of water molecules relaxing under these boundary conditions. Twelve simulations were performed at T = 300 K to study the conformation of the packed molecular system. Bonded and nonbonded interaction potentials were taken from Biosym force fields. The transitions seen in the pi-A curves are explained from the breaks in the plots of potential energy at various concentrations, related to a liquid expanded-to-liquid condensed (LE/LC) transition, and classified under the first order type. Electron diffraction patterns of these films transferred onto transmission electron microscopy (TEM) grids were studied by high-resolution imaging and showed a clear transition from the LE to the LC phase. Copyright 1998 Academic Press.