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Lipase kinetics at the triacylglycerol-water interface using surface tension measurements.

Two methods, the so-called "oil drop" and "Teflon plunger" methods, were designed to monitor lipase hydrolysis of natural long-chain triacylglycerols through the variation with time of the oil-water interfacial tension. The first part of this work is devoted to the development of these two techniques using pure, well-characterized porcine pancreatic lipase. They gave linear responses with enzyme concentrations ranging from 1 x 10(-3) to 30 units x ml-1. We then applied them to a study of the optimal pH conditions for human gastric lipase which were found to range around 5, as previously observed. In the presence of variable concentrations of sodium taurodeoxycholate, these two methods also showed that human gastric lipase is active in the 8-13 dyn cm-1 range of interfacial tension. It is concluded that these two methods, based upon variations with time of the oil-water interfacial tension, constitute reliable, sensitive and convenient means of investigating lipase kinetics.

Animals↗

Sphingomyelin interfacial behavior: the impact of changing acyl chain composition.

Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes.

Biophysical Phenomena↗

Self-assembled shells composed of colloidal particles: fabrication and characterization.

We construct shells with tunable morphology and mechanical response with colloidal particles that self-assemble at the interface of emulsion droplets. Particles self-assemble to minimize the total interfacial energy, spontaneously forming a particle layer that encapsulates the droplets. We stabilize these layers to form solid shells at the droplet interface by aggregating the particles, connecting the particles with adsorbed polymer, or fusing the particles. These techniques reproducibly yield shells with controllable properties such as elastic moduli and breaking forces. To enable diffusive exchange through the particle shells, we transfer them into solvents that are miscible with the encapsulant. We characterize the mechanical properties of the shells by measuring the response to deformation by calibrated microcantilevers.

Colloids↗

Long-range interfacial electron transfer of metalloproteins based on molecular wiring assemblies.

We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a pi-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.

Alcaligenes↗

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol.

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution. Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.

Anisotropy↗

Mechanism of DNA (Southern) and protein (Western) blotting on cellulose nitrate and other membranes.

The transfer of DNA fractions from hydrophilic gels to nitrocellulose membranes (Southern blotting) which was soon followed by the description of an analogous procedure for RNA (Northern blotting), and somewhat later for proteins (Western blotting), has rapidly become an important separation and characterization method in molecular biology, genetic engineering, and immunological detection. Surface tension measurements have shown that the interfacial attraction between DNA and cellulose esters (-delta G132) in aqueous media can be considerable. The weaker binding energy of proteins to cellulose nitrate and to cellulose acetate may be compared to hydrophobic interaction chromatography, as on account of the somewhat lower [-delta G132] values, it often is necessary to "fix" them more tightly onto nitrocellulose by using high salt concentrations. The binding energy of RNA to both cellulose esters also is rather low. In addition to the effect of high ionic strength, the effect of adding methanol, and the effects of denaturation, heating and drying on the energy of attachment of the biopolymers to cellulose esters, have been studied. Cationized nylon membranes have been advocated recently, especially for electrophoretic transfer of nucleic acids (in which process high salt concentrations cannot easily be used). With positively charged nylon membranes, the attachment mainly occurs through the electrostatic attraction between the strongly negatively charged nucleic acids (or proteins) and the positively charged membrane. Also, more apolar membranes (of polyvinyl difluoride) have been proposed, which manifest a strong interfacial (hydrophobic) attraction to all the above biopolymers (regardless of their electrostatic charge). However, with these two novel membrane types it is no longer possible to exploit the large difference in binding energy between DNA and RNA, which makes cellulose nitrate membranes so uniquely suited for RNA-DNA hybridization assays.

Animals↗

Micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free acetone-based, single-bottle primer/adhesives.

OBJECTIVES: The goal of this study was to illustrate and define the micromorphological spectrum which exists at the resin-dentin interface when two water-free, acetone-based, single-bottle primer/adhesive systems (One-Step, Bisco, and Prime & Bond, Dentsply/L.D. Caulk) were applied to acid-conditioned dentin under different dry and wet bonding conditions. METHODS: Forty-eight 1 mm dentin discs were each conditioned with 10% phosphoric acid and rinsed for 20 s. They were randomly divided into 4 groups based upon the status of the remaining surface moisture; Group I (30 s dry); Group II (3 s dry); Group III (blot dry) and Group IV (overwet). Bonded dentin disc pairs were then demineralized in EDTA and embedded in epoxy resin for transmission electron microscopic examination. RESULTS: The micromorphological spectra of the two bonding systems were essentially similar. Both were effected by even mild desiccation (3 s), resulting in incomplete intertubular resin infiltration. Optimal intertubular resin infiltration was achieved when the collagen network was kept moist and appeared relatively unaffected by the presence of excess surface moisture. On the other hand, intratubular resin infiltration was severely compromised in the presence of excess water within the dentinal tubules and at their openings in the dentin surface. The continuity of the resin layer deteriorated; blister-like spaces formed on the dentin surface and resin globules were found around the tubular orifices and on the surface of the hybrid layer. In addition, a complex phase separation pattern was observed in Prime & Bond that was characterized by phase inversion in the presence of surface moisture. SIGNIFICANCE: The "window of opportunity" for optimal interfacial integrity for both water-free systems appeared to depend upon keeping the demineralized collagen network moist, coupled with the complete evaporation of excess and "displaced" water from tissues prior to light-curing the prime/adhesives.

Acetone↗

The lateral pressure profile in membranes: a physical mechanism of general anesthesia.

A mechanism of general anesthesia is suggested and investigated using lattice statistical thermodynamics. Bilayer membranes are characterized by large lateral stresses that vary with depth within the membrane. Incorporation of amphiphilic and other interfacially active solutes into the bilayer is predicted to increase the lateral pressure selectively near the aqueous interfaces, compensated by decreased lateral pressure toward the center of the bilayer. General anesthesia likely involves inhibition of the opening of the ion channel in a postsynaptic ligand-gated membrane protein. If channel opening increases the cross-sectional area of the protein more near the aqueous interface than in the middle of the bilayer, then the anesthetic-induced increase in lateral pressure near the interface will shift the protein conformational equilibrium to favor the closed state, since channel opening will require greater work against this higher pressure. This hypothesis provides a truly mechanistic and thermodynamic understanding of anesthesia, not just correlations of potency with structural or thermodynamic properties. Calculations yield qualitative agreement with anesthetic potency at clinical anesthetic membrane concentrations and predict the alkanol cutoff and anomalously low potencies of strongly hydrophobic molecules with little or no attraction for the aqueous interface, such as perfluorocarbons.

Anesthetics, General↗

Effective kinetic phase diagrams.

The composition of a solid solution that is growing at conditions well away from equilibrium is not prescribed by equilibrium thermodynamics, but is determined kinetically. It depends both on the surface kinetics and on the transport of mass and heat to and away from the solidification front. In previous work, we have formulated a model for the kinetic or nonequilibrium segregation taking place at the solidification front enabling the construction of kinetic phase diagrams, which gives the growth composition of a solid solution as a function of the liquid composition and undercooling at the surface. In the present work, we extend this model to include both mass and heat transport, giving rise to effective kinetic phase diagrams. An overview of the tendencies in the calculated effective kinetic phase diagrams is given by scanning a large part of the parameter space, covering different types of materials, including metals, semiconductors, and molecular systems. We find striking and characteric differences in the relative contribution of the various processes to the effective segregation. For molecular mixtures, interfacial undercooling and heat transport limitation can be expected to be much more important than for metal and semiconductor mixtures where mass transport limitation is dominant.

Journal Article↗

Dynamic finite-size scaling of the normalized height distribution in kinetic surface roughening.

Using well-known simple growth models, we have studied the dynamic finite-size scaling theory for the normalized height distribution of a growing surface. We find a simple functional form that explains size-dependent behavior of the skewness and kurtosis in the transient regime, and obtain the transient- and long-time values of the skewness and kurtosis for the models. Scaled distributions of the models are obtained, and the shape of each distribution is discussed in terms of the interfacial width, skewness, and kurtosis, and compared with those for other models. Exponents eta(+) and eta(-), which characterize the form of the distribution, are determined from an exponential fitting of scaling functions. Our detailed results reveal that eta(+)+eta(-) approximately 4 for a model obeying usual scaling in contrast to eta(+)+eta(-)<4 with eta(-)=1 for a model exhibiting anomalous scaling as well as multiscaling. Since we obtain eta(+)+eta(-) approximately 4 for a model exhibiting anomalous scaling but no multiscaling, we conclude that the deviation from eta(+)+eta(-) approximately 4 is due to the presence of multiscaling behavior in a model.

Journal Article↗

Pivotal role of anionic phospholipids in determining dynamic behavior of lung surfactant.

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are anionic phospholipids (APLs) present in lung surfactant of virtually all species studied, although their specific contribution to function is unknown. This study examines how APLs influence surfactant monolayer stability and adsorption under static and dynamic conditions. Interfacial properties of surfactants reconstituted with native phospholipids (PL), and phospholipids devoid of anionic species (DAPL), were characterized by pulsating bubble surfactometry. Measurements were made for PL and DAPL alone; with 3% surfactant proteins B and C (SP-B/C); with SP-B/C and 5% surfactant protein A (SP-A); and with SP-B/C, SP-A, and 8% neutral lipids (NL). Equilibrium and dynamic properties of PL and DAPL were similar. However, whereas (DAPL + SP-B/C) and (DAPL + SP-B/C + SP-A) mixtures were similar to corresponding PL mixtures with respect to gamma(equil), they displayed markedly different dynamic behavior. In particular, the degree of film compression required to reach gamma(min) was significantly increased in DAPL mixtures (80 to 90% area reduction) compared with PL, although both samples reached gamma(min) < 3.0 dynes/cm. The addition of NL to (DAPL + SP-B/C + SP-A) produced an increase in gamma(min) to 15 to 20 dynes/cm during dynamic compression, whereas NL had no significant impact on the behavior of (PL + SP-B/C + SP-A). Purified PG (5% wt/wt) restored nearly normal dynamic properties to (DAPL + SP-B/C + SP-A + NL), whereas phosphatidylcholine (PC) (5% wt/wt) had no beneficial effect. These results suggest that APLs play a critical role in promoting surface film stability during dynamic compression through interactions with nonlipid surfactant components, and prevent destabilization of the surface film by cholesterol and other NL.

Animals↗

Membrane fluidity gradient model of cell transport.

A new model of cellular transport is presented, characterized by selective fluxes due to membrane fluidity gradient. This mechanism is treated in terms of the interfacial tensions at the membrane/cytoplasm and membrane/medium surfaces. A higher interior fluidity (lower interfacial tension) is maintained by cytoplasm adenosine triphosphate, which adsorbs and increases lipoprotein fluidity while it also chelates calcium and keeps it from inner membrane sites. The high medium calcium causes a stiffer membrane (higher interfacial tension) on the medium side. These two different free energy barriers at inner and outer channel mouths filter all molecules, whether ionized or nonelectrolytic. Molecules with excess of hydrophobic groups, which makes negative the free energy of transfer from the medium into the membrane, have highest influx. Intermolecular salt linkages and hydrogen-bonding are vital in making negative the free energy of transfer of amino acids and sugars. The much lower energy barrier at the cytoplasmic interface favors net efflux from the cell of the more polar ions and amphipaths. Intramembrane particles are proposed as the channel sites.

Adenosine Triphosphate↗

Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins.

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins.

Bacterial Proteins↗

Using O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates to investigate the role of Ca2+ and interfacial binding in a bacterial phospholipase D.

O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates (n=6, 8, and 10; CnPNC) were synthesized and characterized as inhibitors of phospholipase D (PLD) activity toward phosphatidylcholine presented as monomers, micelles, and bilayers. Detailed studies with recombinant Streptomyces chromofuscus PLD, a Ca(2+)-activated enzyme that does not show large changes in catalytic activity toward the same substrate as a monomer or micelle, showed that the longer the inhibitor chain length, the more potent CnPNC is as a competitive inhibitor toward all the substrates. However, the physical state of the inhibitor did affect the maximum inhibition attainable. For a fixed concentration of diC4PC (monomer substrate), CnPNC inhibition reached a maximum around the CMC of the inhibitor; the inhibition was reduced at higher inhibitor concentrations, in part caused by the lower solubility of the aggregated inhibitor. With diC4PC as the substrate and using concentrations of C10PNC that were below its CMC, the Ki for C10PNC was 0.030+/-0.003 mM, approximately 13-fold less than the Km for substrate. Aggregated substrates showed significant inhibition of PLD by CnPNC, although as the substrate chain length increased, inhibition by a given CnPNC was diminished. With POPC vesicles, the apparent Ki for C10PNC was 0.030 of the apparent Km. The availability of these inhibitors allowed us to show that PC analogues can bind to the active site of S. chromofuscus PLD in the absence of Ca2+. Once bound at the active site, the inhibitor does not significantly affect the divalent ion-dependent partitioning of the enzyme to PC surfaces. Of the two other PLD enzymes examined, cabbage PLD, but not Streptomyces sp. PMF, was able to catalyze the cleavage of the P-N bond. Differential susceptibility of PLDs to these phosphoramidates may eventually be useful in studying PLD isozymes in cells.

Amides↗

Intrinsic molecules in lipid membranes change the lipid-domain interfacial area: cholesterol at domain interfaces.

A theoretical analysis of the effects of intrinsic molecules on the lateral density fluctuations in lipid bilayer membranes is carried out by means of computer simulations on a microscopic interaction model of the gel-to-fluid chain-melting phase transition. The inhomogeneous equilibrium structures of gel and fluid domains, which in previous work (Cruzeiro-Hansson, L. and Mouritsen, O.G. (1988) Biochim. Biophys. Acta 944, 63-72) were shown to characterize the transition region of pure lipid membranes, are here shown to be enhanced by intrinsic molecules such as cholesterol. Cholesterol is found to increase the interfacial area and to accumulate in the interfaces. The interfacial area, the average cluster size, the lateral compressibility, and the membrane area are calculated as functions of temperature and cholesterol concentration. It is shown that the enhancement by cholesterol of the lateral density fluctuations and the lipid-domain interfacial area is most pronounced away from the transition temperature. The implications of the results are discussed in relation to passive ion permeability and function of interfacially active enzymes such as phospholipase.

Cell Membrane Permeability↗

Effect of pre-strain on interfacial friction damping in carbon nanotube polymer composites.

This paper investigates the effect of mechanical pre-strain on interfacial friction damping in nanotube polymer composites. Oxidized single-walled carbon nanotubes were dispersed in a polycarbonate matrix using a solution mixing technique. To characterize the damping response, the material storage and loss modulus was measured by application of dynamic (sinusoidal) load to the nanocomposite in the uniaxial direction. A static pre-strain (in 0.35-0.85% range) was then superimposed on the dynamic strain to quantify its effect on the material response. The results indicate that application of pre-strain facilitates the activation of interfacial slip at the nanotube-polymer interfaces at relatively low dynamic strain amplitudes. This is because pre-strain raises the interfacial shear stress for the nanotube inclusions allowing the critical stress for tube-matrix interfacial slip to be reached at lower strain amplitudes. In this way pre-strain significantly improves the effectiveness of the nanotube-matrix sliding energy dissipation mechanism for damping enhancement in composite structures.

Carbon↗

Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues.

The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.

Fluorescent Dyes↗

Molecular adsorption at silica/CH3CN interface probed by using evanescent wave cavity ring-down absorption spectroscopy: determination of thermodynamic properties.

Evanescent wave cavity ring-down absorption spectroscopy is applied to measure the thermodynamic properties of the surface adsorption for neutral trans-4-[4-(dibutylamino)styryl]-1-(3-sulfopropyl) pyridinium (DP) and charged trans-4-[4-(dibutylamino)styryl]-1-methylpyridinium iodide (DMP+ I-) at the silica/CH3CN interface, where the interfacial density is determined by measurement of absorbance. The bulk concentration dependence of the surface density may be characterized with a Langmuir isotherm model, which yields saturated surface density, equilibrium constant, and free energy of adsorption of (7.0 +/- 0.3) x 10(13) cm(-2), (1.3 +/- 0.2) x 10(4) M(-1), and -23.5 +/- 0.4 kJ/mol for DP and (8.9 +/- 0.3) x 10(12) cm(-2), (2.6 +/- 0.7) x 10(4) M(-1), and -25.2 +/- 0.6 kJ/mol for DMP+ I-, respectively. The surface density of the isolated silanol groups may then be estimated in terms of the molecular probe results. The absorption contribution from the bulk solution is a factor of approximately 10(1)-10(2) smaller than the total absorbance measured such that subtraction of the bulk contribution leads to negligible change of the thermodynamic properties. The DP is adsorbed to the SiOH sites by forming hydrogen bonds, while the DMP+ cation is bound to the SiO- sites by electrostatic attraction. Surface forces are also probed by addition of triethylamine (TEA), which is competitive with DP for the silanol sites. When the TEA concentration is increased, the DP surface density is found to decrease, whereas the DMP+ surface density increases. The obtained thermodynamic properties are generally consistent with those measured by second harmonic generation spectroscopy. However, when a tetramethylammonium ((CH3)4N+ Cl-) salt is added, the DMP+ cation behaves differently between these two methods. Formation of an electrical double layer may account for the difference.

Journal Article↗