Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein on phospholipid Langmuir monolayers

The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.     

Enzymatic hydrolysis reaction of phospholipids in monolayers

The hydrolysis reaction of , and , -dipalmitoylphosphatidylcholine (DPPC) catalized by bee venom phospholipase A₂ was studied in spreading monolayer at the water/air interface. DPPC and the hydrolysis products, palmitic acid and -lysophosphatidylcholine, palmitoyl were characterized at the interface by means of surface pressure, surface potential and ellipsometric measurements. Furthermore, mixed monolayers of reagents and products were investigated to ascertain their miscibility. The results show that the hydrolysis reaction can be followed by the decrease of surface pressure with time on subphases containing β-cyclodextrin, a well-known complexing agent of many amphiphilic compounds. The order of the reaction, the kinetic constant and other kinetic parameters are deduced.     

Nanomolar protein sensing with embedded receptor molecules

A new concept of protein sensing at the air-water interface is introduced, based on amphiphilic receptor molecules embedded in a lipid monolayer. The process begins with incorporation of a small amount (0.13 equiv) of one or two different calix[4]arenes, adorned with charged functional groups at their upper rims, into a stearic acid monolayer. These doped monolayers are subsequently shown to attract peptides and proteins from the aqueous subphase. Depending on the host structure, the monolayers can be made selective for basic or acidic proteins. A working model is proposed, which explains the large observed p/A shifts with reincorporation of excess receptor molecules into the lipid monolayer after complex formation with the oppositely charged protein. This requires a self-assembly of multiple calixarene units over the protein surface, which bind the protein in a cooperative fashion. Oppositely charged calixarene derivatives do not form molecular capsules inside the monolayer, but rather remain separate inside the lipid layer, adopting a perpendicular orientation. They combine their hydrogen bond donor and acceptor capacities, and thus markedly enhance the sensitivity of the sensor system toward proteins, pushing the detection limits to 10 pM concentrations. The response pattern obtained from various receptor units inside the monolayer toward the same protein creates a fingerprint for this protein, which can hence be selectively detected at nanomolar concentrations (pattern recognition).     

Two-dimensional arrays of amphiphilic Zn2+-cyclens for guided molecular recognition at interfaces

The sterically guided molecular recognition of nucleobases, phosphates, adenosine, and uridine nucleotides on Langmuir monolayers and Langmuir-Blodgett monolayers of amphiphilic mono- or bis(Zn2+-cyclen)s assembled on thiolated surfaces was investigated. The stepwise selective binding of metal ions, uracil, or phosphate by dicetyl cyclen monolayers with variously tuned structures at the air/water interface was corroborated by the measurements of the corresponding LB films deposited onto quartz crystals. Two types of recognition surfaces were fabricated from Zn2+-dicetyl cyclen. The surface covered with a complex preformed in the Langmuir monolayer was capable both of imide and of phosphate binding. The similar complex formed directly in an LB film on thiolated gold was inactive with respect to imide. The surface plasmon resonance measurements evidenced the stepwise assembly of complementary nucleotides on SAM/LB templates through consecutive phosphate-Zn2+-cyclen coordination. Base pairing between nucleotides resulted in a formation of A-U bilayers comprising two complementary monolayers. Finally, we report on SAM/LB patterns designed for divalent molecular recognition of uridine phosphate by amphiphilic bis(Zn2+-cyclen).     

Fluorescence transduction of an enzyme-substrate reaction by modulation of lipid membrane structure

The emission intensity of the fluorophore nitrobenzoxadiazoledipalmitoylphosphatidylethanolamine (NBD-PE) is sensitive to local environmental structure when this species is used as a component of a phospholipid membrane. The physical and electrostatic structure of a membrane may be modulated by selective chemical reactions, and the resulting alteration in fluorescence intensity provides transduction of such selective chemical processes. One example is the reaction between the extrinsic membrane-associated enzyme acetylcholinesterase (AChE) and the substrate acetylcholine (ACh), which produces an increase in hydronium ion activity at the surface of a lipid membrane. A mechanism of transduction of the enzymatic reaction by lipid monolayer membranes was investigated by spectrofluorimetric methods and fluorescence microscopy. Mixed monolayers composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) which contained 30 mol-% or more of DPPA and 1 mol-% of NBD-PE provided transduction of the AChEACh reaction. Reaction of micromolar concentrations of ACh with AChE-monolayer systems induced increases in fluorescence intensity of up to 50%. Direct observation of the microscopic structure of lipid monolayers on a time scale of minutes showed that the reaction did not drastically affect the distribution of coexisting microscopic phase domains that were present in the monolayers The fluorescence imaging and spectroscopic results did indicate that massive structural reorganization at a molecular level probably occurred in a period of seconds. The results are consistent with an electrostatic mechanism of perturbation of the structure of the monolayer in which local pH gradients associated with the reaction of AChE with substrate altered the extent of ionization of DPPA in the headgroup zone of the membrane.     

Molecular organization of a water-insoluble iridium(III) complex in mixed monolayers

In this work, organized mixed monolayers containing a cationic water-insoluble iridium(III) complex, Ir-dye, [Ir(ppy)(2)(tmphen)]PF(6), (tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline, and ppy = 2-phenylpyridine), and an anionic lipid matrix, DMPA, dimyristoyl-phosphatidic acid, with different molar proportions, were formed by the co-spreading method at the air-water interface. The presence of the dye at the interface, as well as the molecular organization of the mixed films, is deduced from surface techniques such as pi-A isotherms, Brewster angle microscopy (BAM) and reflection spectroscopy. The results obtained remark the formation of an equimolar mixed film, Ir-dye/DMPA = 1:1. BAM images reveal a whole homogeneous monolayer, with gradually increasing reflectivity along the compression process up to reaching the collapse of this equimolecular monolayer at pi approximately equal to 37 mNm(-1). Increasing the molar ratio of DMPA in the mixture, the excess of lipid molecules organizes themselves forming dark flower-like domains of pure DMPA at high surface pressures, coexisting with the mixed Ir-dye/DMPA = 1:1 monolayer. On the other hand, unstable mixed monolayers are obtained by using an initial dye surface concentration higher than the equimolecular one. These mixed Langmuir monolayers have been successfully transferred onto solid substrates by the LB (Langmuir-Blodgett) technique.     

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.     

Interfacially formed organized planar inorganic, polymeric and composite nanostructures

This paper discusses synthetic strategies for fabrication of new organized planar inorganic, polymeric, composite and bio-inorganic nanostructures by methods based on chemical reactions and physical interactions at the gas-liquid interface, Langmuir monolayer technique, interfacial ligand exchange and substitution reactions, self-assembling and self-organization processes, DNA templating and scaffolding. Stable reproducible planar assemblies of ligand-stabilized molecular nanoclusters containing definite number of atoms have been formed on solid substrate surfaces via preparation and deposition of mixed Langmuir monolayers composed by nanocluster and surfactant molecules. A novel approach to synthesis of inorganic nanoparticles and to formation of self-organized planar inorganic nanostructures has been introduced. In that approach, nanoparticles and nanostructures are fabricated via decomposition of insoluble metal-organic precursor compounds in a layer at the gas-liquid interface. The ultimately thin and anisotropic dynamic monomolecular reaction system was realized in that approach with quasi-two-dimensional growth and organization of nanoparticles and nanostructures in the plain of Langmuir monolayer. Photochemical and redox reactions were used to initiate processes of interfacial nucleation and growth of inorganic phase. It has been demonstrated that morphology of resulting inorganic nanostructures can be controlled efficiently by variations of growth conditions via changes in state and composition of interfacial planar reaction media, and by variations of composition of adjacent bulk phases. Planar arrays and chains of iron oxide and ultrasmall noble metal (Au and Pd) nanoparticles, nanowires and new organized planar disk, ring, net-like, labyrinth and very high-surface area nanostructures were obtained by methods based on that approach. Highly organized monomolecular polymeric films on solid substrates were obtained via deposition of Langmuir monolayer formed by water-insoluble amphiphilic polycation molecules. Corresponding nanoscale-ordered planar polymeric nanocomposite films with incorporated ligand-stabilized molecular metallic nanoclusters and interfacially grown nanoparticles were fabricated successfully. Novel planar DNA complexes with amphiphilic polycation monolayer were formed at the gas-aqueous phase interface and then deposited on solid substrates. Toroidal and new net-like conformations were discovered in those complexes. Nanoscale supramolecular organization of the complexes was dependent on cationic amphiphile monolayer state during the DNA binding. These monolayer and multilayer DNA/amphiphilic polycation complex Langmuir-Blodgett films were used as templates and nanoreactors for generation of inorganic nanostructures via metal cation binding with DNA and following inorganic phase growth reactions. As a result, ultrathin polymeric nanocomposite films with integrated DNA building blocks and organized inorganic semiconductor (CdS) and iron oxide quasi-linear nanostructures were formed. It has been demonstrated that interaction of deposited planar DNA/amphiphilic polycation complexes with bulk phase colloid inorganic cationic ligands (CdSe nano-rods) can result in formation of new highly organized hybrid bio-inorganic nanostructures via interfacial ligand exchange and self-organization processes. The methods developed can be useful for investigation of fundamental mechanisms of nanoscale structural organization and transformation processes in various inorganic and molecular systems including bio-molecular and bio-inorganic nanostructures. Also, those methods are relatively simple, environmentally safe and thus could prove to be efficient practical instruments of molecular nanotechnology with potential of design and cost-effective fabrication of new controlled-morphology organized planar inorganic and composite nanostructured materials. Possible applications of obtained nanostructures and future developments are also discussed.     

Hydrolysis characterization of phospholipid monolayers catalyzed by different phospholipases at the air-water interface

Combination of some newly developed microscopic and spectroscopic techniques with conventional Langmuir monolayer method can provide more quantitative information with the molecular orientation and reorganization process of spread amphiphilic molecules at the air/water interface. These techniques are extended to investigate the hydrolysis process of spreading lipid monolayer catalyzed by different enzymes, phospholipases A2, C and D, respectively. Synchrotron X-ray diffraction and infrared reflection absorption spectroscopy are able directly to give the structural information of the assembled monolayer, interfacial activity of amphiphiles and their components at the interface. Microscopic technique such as Brewster angle microscopy (BAM), fluorescence microscopy (FM) can be used to trace the morphological changes dynamically as the spreading lipid monolayer is hydrolyzed at the air/water interface. We summary here some latest progress in this filed and give a brief review over the hydrolysis features of phospholipid monolayer catalyzed by different enzymes. It is attempted to establish a model of membrane hydrolysis process in order to better understand the mechanism of membrane metabolism and signal transduction in a living system.     

Discreteness-of-charge-effects in the shift of the metallation equilibrium of an amphiphilic porphyrin in a lipid monolayer

Summary The metallation equilibrium of an amphiphilic porphyrin in a mixed lipid monolayer may be changed at the air/water interface, independent of the overall surface potential. Langmuir-Blodgett assemblies of the same lipid monolayer are used to prove that the mean surface potential only shifts the porphyrins' protonation equilibrium, but not its metallation equilibrium. It is then shown that the choice of a cationic porphyrin embedded in a fatty acid lipid matrix (dominating the surface potential) allows the metallation equilibrium to be shifted by more than 4 pK-units as compared to the same system with either an electrically neutral porphyrin or lipid matrix. This is due to a molecular order in the mixed monolayer with peripheral positive charges dissociating the free base porphyrin and the fatty acid molecules sitting on top of the porphyrins' chromophores, facilitating the incorporation of a metalion. Evidence for this molecular arrangement is given by means of -A-Isotherms and reflection spectroscopy.     

Phospholipid morphologies on photochemically patterned silane monolayers

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.     

Mixed DPPC/DPTAP monolayers at the air/water interface: influence of indolilo-3-acetic acid and selenate ions on the monolayer morphology

The interactions of mixed monolayers of two lipids, zwitterionic 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), with phytohormone indolilo-3-acetic acid (IAA) and selenate anions in the aqueous subphase were studied. For this purpose, isotherms of the surface pressure versus the mean molecular area were recorded. Domain formation was investigated by using Brewster angle microscopy (BAM). The method of grazing incidence X-ray diffraction (GIXD) was also applied for the characterization of the organization of lipid molecules in condensed monolayers. It was found that selenate ions contribute to monolayer condensation by neutralizing the positive net charge of mixed monolayers whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization. When both solutes were introduced into the subphase, a competition between them for interaction with the positively charged lipids in the monolayer was observed.     

Nucleotide recognition and phosphate linkage hydrolysis at a lipid cubic interface

Mononucleotides, when entrapped within a mono-olein-based cubic Ia3d liquid crystalline phase, have been found to undergo hydrolysis at the sugar-phosphate ester bond in spite of their natural inertness toward hydrolysis. Here, kinetics of the hydrolysis reaction and interactions between the lipid matrix and the mononucleotide adenosine 5'-monophosphate disodium salt (AMP) and its 2'-deoxy derivative (dAMP) are thoroughly investigated in order to shed some light on the mechanism of the nucleotide recognition and phosphate ester hydrolysis. Experiments evidenced that molecular recognition occurs essentially through the sn-2 and the sn-3 alcoholic OH groups of mono-olein. As deduced from the apparent activation energies, the mechanism underlying the hydrolysis reaction is the same for AMP and dAMP. Nevertheless, the reaction proceeds slower for the latter, highlighting a substantial difference in the chemical behavior of the two nucleotides. A model that explains the hydrolysis reaction is presented. Remarkably, the hydrolysis mechanism appears to be highly specific for the Ia3d phase.     

Structural characteristics of dipalmitoylphosphatidylcholine and hydrolysates from sunflower protein isolate mixed monolayers spread at the air-water interface

In this work, surface film balance and Brewster angle microscopy techniques have been used to analyze the structural characteristics (structure, topography, reflectivity, thickness, miscibility, and interactions) of hydrolysates from sunflower protein isolate (SPI) and dipalmitoylphosphatidylcholine (DPPC) mixed monolayers spread on the air-water interface. The degree of hydrolysis (DH) of SPI, low (5.62%), medium (23.5%), and high (46.3%), and the protein/DPPC mass fraction were analyzed as variables. The structural characteristics of the mixed monolayers deduced from the surface pressure (pi)-area (A) isotherms depend on the interfacial composition and degree of hydrolysis. At surface pressures lower than the equilibrium surface pressure of SPI hydrolysate (pi(e)(SPI hydrolysate)), both DPPC and protein are present in the mixed monolayer. At higher surface pressures (at pi > pi(e)(SPI hydrolysate)), collapsed protein residues may be displaced from the interface by DPPC molecules. The differences observed between pure SPI hydrolysates and DPPC in reflectivity (I) and monolayer thickness during monolayer compression have been used to analyze the topographical characteristics of SPI hydrolysates and DPPC mixed monolayers at the air-water interface. The topography, reflectivity, and thickness of mixed monolayers confirm at microscopic and nanoscopic levels the structural characteristics deduced from the pi-A isotherms.     

In situ determination of orientational distributions in Langmuir monolayers by total internal reflection fluorescence

A new application of the polarized total internal reflection fluorescence (PTIRF) technique to study the orientation distribution of a fluorophore within a Langmuir monolayer in situ on an aqueous subphase is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The excitation by the evanescent field is achieved by launching the beam into a prism that is brought into contact with the monolayer from above. We also show here that a combination of PTIRF of monolayers on water and those freshly deposited onto the prism by horizontal lift in the same experiment provide enough data to determine the dielectric constant of the actual local environment of the fluorophore in the monolayer to eliminate the ambiguity of the orientation determination, arising from uncertainty in the normal component of excitation field. The new technique was applied to several model systems: fatty acid monolayers containing amphiphilic dyes DiI or BODIPY and also a monolayer of a synthetic amphiphilic porphyrin-binding peptide AP0. This technique is more accurate than polarized epifluorescence (PEF) in determining the fluorophore orientation distribution due to the much higher normal component of the excitation, achievable in the evanescent field, and to the lack of surface vibrations caused by capillary waves. Comparison of the new PTIRF approach with PEF shows that the monolayer structure is not disturbed by weak van der Waals attachment to the hydrophobic substrate.     

Effects of lipid chain length on the surface properties of alkylaminomethyl rutin and of its mixture with model lecithin membrane

Three model flavonoid-based bioactive molecules with different lipid chain lengths (RuCn: n=8, 12, 18) were newly synthesized. The surface properties [surface pressure (π)-area (A), surface potential (ΔV)-surface pressure (π) and dipole moment (u(⊥))-surface pressure (π)] of pure RuCn and the lecithin membrane compounds had been investigated by using the Langmuir monolayer technology. The results suggested that the distinctive monolayer behavior of RuCn is strongly dependent on the lipid chain length. The great differences in the monolayer properties brought by the lipid chain length could be attributed to two major factors: (i) the ionization degree of the bulky hydrophilic head group (including hydroxyl and NH groups) alters its local field solely via the surface potential; (ii) tring molecular (or dipole) packing density within monolayers. The excess Gibbs energy (ΔG((ex))) calculated for the RuCn-lecithin mixed monolayers infers that higher stability of the mixed monolayer can be strengthened as the lipid chain length decreases. And the addition of RuCn into lecithin membrane may increase the total u(⊥) of the binary mixed monolayers, which could inhibit the hydration of the lecithin's hydrophilic head groups. The shorter the lipid chain length of RuCn (e.g., RuC8) is, the higher the surface activity can be. Our findings provide a molecular basis for the application of such class of biomolecules in the functional food, cosmetics and medicine.     

Neutral Nickel(II) and Copper(II) Tetraazamacrocyclic Complexes as Molecular Rods Attached to Gold Electrodes

New dithiolated derivatives of neutral Cu^II and Ni^II tetraazamacrocyclic complexes have been synthesized and characterized by spectroscopic and diffractional methods. These rod‐shaped molecules were assembled in monocomponent and mixed monolayers on gold electrodes. In the mixed monolayers, the active molecules were embedded in a hexanethiol matrix. The dithiolated complexes are oriented perpendicularly to the electrode, and reveal faster kinetics of electron transfer than those assembled in a single‐component monolayer. They appear as protrusions, which are easily addressed by using the STM method. In the presence of a suitable electron acceptor in the solution, the donor properties of the anchored Cu complex were weakened, which revealed donor–acceptor interactions with the monolayer. The peak position in the voltammogram indicates a stronger interaction of the solution‐based acceptor with the reduced Cu^II form than with the Cu^III complex. This suggests the possibility of switching the association on or off by applying an appropriate potential.     

Simplified speciation and improved phosphodiesterolytic activity of hydroxo complexes of trivalent lanthanides in aqueous DMSO

Potentiometric titrations of La(III), Nd(III), and Eu(III) perchlorates by Me 4N(OH) in 80% vol aq DMSO revealed formation of predominantly mononuclear complexes M(OH)n(3- n) (n = 1, 2, or 3) and a single binuclear complex M2(OH)(5+). Kinetics of the cleavage of two phosphate diesters, bis (4-nitrophenyl) phosphate (BNPP) and 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP), and a triester, 4-nitrophenyl diethyl phosphate (paraoxon), were studied as a function of metal and Me4N(OH) concentrations in the same medium. Rate of BNPP cleavage is second-order in metal and is proportional to the product of concentrations of M(OH)2(+) and M(OH)3 species. Rate of HPNPP cleavage is proportional to [M(OH)3](3) for La(III) and Nd(III) and to [M(OH)3](2) for Eu(III). Proposed mechanism for BNPP hydrolysis involves formation of M2(OH)5(diester) intermediate followed by intramolecular nucleophilic attack of hydroxide anion on the phosphoryl group of the substrate. Proposed mechanism for HPNPP cleavage involves formation of M3(OH)9(diester)(-) or M2(OH)6(diester)(-) intermediates followed by the general base-assisted intramolecular cyclization of HPNPP. The latter mechanism is supported by observation of the solvent kinetic isotope effect k H/kD = 2.9 for Eu(III) catalyzed HPNPP cleavage. The efficiency of catalysis in 80% DMSO is much higher than in water. The reaction rate observed in the presence of 1 mM metal in neutral solution surpasses the rate of background hydrolysis by a factor of 10(12)-10(13) for BNPP and 10(10) for HPNPP. The increased catalytic activity is attributed principally to the preferable solvation of lanthanide ions by DMSO, which creates an anhydrous microenvironment favorable for reaction in the coordination sphere of the catalyst. The catalytic activity of lanthanides in paraoxon hydrolysis is much lower with the estimated efficiency of catalysis about 10(5) for 1 mM La(III).