Radha Ranganathan

Aggregate Properties of Sodium Deoxycholate and Dimyristoylphosphatidylcholine Mixed Micelles

Mixed micelles of the phospholipid dimyristoylphosphatidylcholine (DMPC) and bile salts of sodium deoxycholate (NaDC) were investigated by a combination of techniques, including time-resolved fluorescence quenching (TRFQ), electron spin resonance (ESR), viscometry, pulsed-gradient spin-echo NMR (PGSE-NMR), and surface tensiometry. Aggregation numbers, and bimolecular collision rate constants of guest molecules confined in the micelles (by TRFQ), interfacial hydration index and microviscosity, (by ESR), axial ratio (from solution viscosity), micelle self-diffusion coefficient (by PGSE-NMR), and the critical micelle concentrations (from surface tension) were determined for various molar compositions defined by the ratio R identical with [NaDC]/[DMPC] and concentrations ([NaDC]+[DMPC]). The data interpretation showed the micelles to be polydisperse rods. Aggregate properties depend on the ratio, R and reveal behavior unlike that in micelles of surfactants with aliphatic nonpolar chains. With increase in concentration from [NaDC] = 0.010 M to [NaDC] = 0.200 M, the hydration index and the aggregation number exhibit non-monotonic variations. Formulation of a polar shell model for cylindrical micelles yielded a set of nonlinear equations for the structural features of the micelle. The solutions give the microstructural description of the mixed micelle that includes the length, diameter, number of water molecules in the hydration shell, and the monomer organization in the micelle.

Amphiphilic lauryl ester derivatives from aromatic amino acids: significance of chemical architecture in aqueous aggregation properties.

Lauryl esters of L-tyrosine (LET) and L-phenylalanine (LEP) were, in a previous interface adsorption study, found to adopt very different interfacial conformations. The present study is an investigation of their aqueous aggregation properties with the goal of elucidating the effects of the presence in LET and absence in LEP of the phenolic OH group on their aqueous aggregate structures and micellar conformations of the surfactant monomers. The measured properties included aggregation numbers from time-resolved fluorescence quenching (TRFQ), interface hydration index and microviscosity by electron spin resonance (ESR), chemical shifts of (1)H resonance lines by NMR, and Krafft temperatures and enthalpies of structural transitions by differential scanning calorimetry (DSC). The TRFQ, ESR, and NMR experiments were conducted at various temperatures from 23 to 70 degrees C for various surfactant concentrations from 0.050 to 0.200 M. Markedly different temperature dependences of aggregation number and (1)H NMR chemical shifts are exhibited by LET and LEP micelles. LET and LEP form ionic micelles. The aggregation number of LEP decreases as is characteristic of ionic micelles, but that of LET increases slightly with temperature. The changes with temperature in the NMR chemical shifts and width of the resonance lines are significantly greater for the various LEP protons than for those of LET. The differences in these properties and other fluorescence decay characteristics of fluorophores incorporated into the micelles could be attributed to the difference in the micellar conformations of LET and LEP which are postulated to be similar to that at oil-water interfaces. The phenolic group is hypothesized to be in the micelle-water interface as part of the headgroup in LET micelles, and its location does not change with temperature. On the other hand, in LEP micelles, the phenyl ring is folded into the core overlapping with the flexible hydrophobic chains. The resulting closer proximity between the phenyl ring and the flexible hydrocarbon chain causes interdependence of the phenyl ring and chain proton resonances, leading to the observed temperature dependence of the chemical shifts in LEP. The TRFQ and ESR data are combined together in a molecular space-filling model, referred to as the polar shell model, to derive the geometrical properties of the micelle. The DSC scans in the temperature range 10-55 degrees C showed the presence of distinctly different endotherms for LET and LEP. The Krafft temperatures, K(T), and the enthalpies were determined. The higher K(T) and broader peak of the DSC endotherm of LET as compared to LEP are attributed to the stabilization of fiberlike structures below the Krafft temperature due to its chirality and the hydrogen bonding capability of the phenolic OH and also to the ion-dipole interactions. Thus, all of the observed differences between LET and LEP could be attributed to the difference in their chemical architecture.

Surface dilution kinetics using substrate analog enantiomers as diluents: enzymatic lipolysis by bee venom phospholipase A2.

A novel assay employing D-enantiomers of phospholipids as diluents for characterizing surface kinetics of lipid hydrolysis by phospholipases is introduced. The rationales of the method are (i) D-enantiomers resist hydrolysis because of the stereoselectivity of the enzymes toward L-enantiomers and (ii) mixtures of L+D-lipids at various L/D ratios but constant L+D-lipid concentrations yield a surface dilution series of variable L-lipid concentration with constant medium properties. Kinetic characterization of bee venom phospholipase A(2) activity at bile salt+phospholipid aggregate-water interfaces was performed using the mixed L+D-lipid surface dilution assay, and interface kinetic parameters were obtained. The assay applies to biomembrane models as well. Activity was measured by pH-stat methods. Aggregation numbers and interface hydration/microviscosity measured by time-resolved fluorescence quenching and electron spin resonance, respectively, confirmed that interface properties were indeed invariant in a surface dilution series, supporting rationale (ii), and were used to calculate substrate concentrations. Activity data show excellent agreement with a kinetic model derived with D-enantiomers as diluents and also that D-phospholipids bind to the enzyme but resist hydrolysis; underscoring rationale (i). The assay is significant for enabling determination of interface-specific kinetic parameters for the first time and thereby characterization of interface specificity of lipolytic enzymes.

Surface dilution kinetics of phospholipase A(2) catalyzed lipid-bilayer hydrolysis.

Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of phospholipids in membranes. Elucidation of the kinetics of interfacial enzymatic activity is best accomplished by investigating the interface substrate concentration dependence of the activity for which appropriate diluents are required. PLA2 is stereoselective toward the L_enantiomers of phospholipids. A novel approach employing D_phospholipids as diluents to perform surface dilution kinetic studies of PLA2 is presented. Activity of bee venom PLA2 at mixed L+D_DPPC (dipalmitoylphosphatidylcholine) bilayer interfaces was measured as a function of substrate L_DPPC mole fraction and vesicle concentration using a sensitive fluorescence assay. A model for interface enzymatic activity based on the three-step kinetic scheme of (i) binding of PLA2 to the bilayer interface, (ii) binding of a lipid to PLA2 at the interface, and (iii) hydrolysis was applied to the hydrolysis data. Activity profiles showed that D_enantiomers also bind to the enzyme but resist hydrolysis. Activity dependences on vesicle and substrate concentrations could be disentangled, bringing resolution to an outstanding problem in membrane hydrolysis of separating the effects of the three steps. Individual values of the kinetic parameters of the model, including the vesicle-PLA2 equilibrium dissociation constant of step (i), interface Michaelis-Menten-Henri constant for L and D_DPPC of step (ii), and the rate constant for interface hydrolysis, step (iii), were obtained as solutions to equations resulting from fitting the model to the data.

Synthesis of mixed-chain phosphatidylcholines including coumarin fluorophores for FRET-based kinetic studies of phospholipase A 2 enzymes

Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester linkage of glycerophospholipids to produce fatty acids and lysophospholipids. A significant number of mammalian phospholipases comprise a family of secreted PLA2 enzymes, found in specific tissues and cellular locations, exhibiting unique enzymatic properties and distinct biological functions. Development of new real-time spectrofluorimetric PLA2 assays should facilitate the kinetic characterization and mechanistic elucidation of the isozymes in vitro, with the potential applicability to detect and measure catalytic PLA2 activity in tissues and cellular locations. Here we report a new synthesis of double-labeled phosphatidylcholine analogs with chain-terminal reporter groups including coumarin fluorophores for fluorescence resonance energy transfer (FRET)-based kinetic studies of PLA2 enzymes. The use of coumarin derivatives as fluorescent labels provides reporter groups with substantially decreased size compared to the first generation of donor-acceptor pairs of fluorescent phospholipids. The key advantage of the design is to interfere less with the physicochemical properties of the acyl chains, thereby improving the substrate quality of the synthetic probes. In order to assess the impact of the fluorophore substituents on the catalytic hydrolysis and on the phospholipid packing in the lipid-water interface of the assay, we used the experimentally determined specific activity of bee-venom phospholipase A2 as a model for the secretory PLA2 enzymes. Specifically, the rate of PLA2 hydrolysis of the coumarin labeled phosphatidylcholine analogs was less than three times slower than the natural substrate dipalmitoyl phosphatidylcholine (DPPC) under the same experimental conditions. Furthermore, variation of the mole fraction of the synthetic phosphatidylcholine vs. that of the natural DPPC substrate showed nearly ideal mixing behavior in the phospholipid-surfactant aggregates of the assay. The synthesis provides a rapid and efficient method for preparation of new synthetic phosphatidylcholines with the desired target structures for enzymatic and physicochemical studies.

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA. Binding displaces Acrylodan and its fluorescence peak shifts from 432 to 505 nm. A fluorescence assay that relies on this shift is presented for quantitating LPC, FA, and phospholipase A2 (PLA2) activity in phospholipid bilayers in absolute units of μM/min/mg of enzyme. This is a development over an earlier assay that took into account only FA binding. Activities of bee venom PLA2 on dipalmitoylphosphatidylcholine (DPPC) and dioleylphosphatidylcholine (DOPC) bilayers were measured. Standard pH-Stat assays validated the present assay. Products increase linearly with time for about one minute in DOPC and five minutes in DPPC corresponding to completion of 5 to 8% hydrolysis in DOPC and 20% in DPPC. Membrane polarity and microviscosity measured using electron spin resonance (ESR) exhibited discontinuities at compositions that mimicked similar percentages of hydrolysis products in the respective bilayers. The observed hydrolysis rate decrease following the initial linear period thus correlates to changes in membrane polarity. The ability of the assay to yield actual product concentrations, reveal structure in the reaction progress curves, and interpretation in light of the ESR data bring insight into the shape of the reaction curve.

pH-responsive aggregation states of chiral polymerizable amphiphiles from L-tyrosine and L-phenyl alanine in water.

Sodium salts of maleamic acid derivatives of lauryl ester of tyrosine (MTNa) and phenyl alanine (MPNa) in water exhibited strong pH-responsive behaviors of viscosity and specific conductivity that originate from the concentration and pH dependence of their aggregation states. The aggregates were characterized by a novel spin-probe-partitioning electron paramagnetic resonance (SPPEPR) method and dynamic light scattering (DLS). Results of high-precision fitting of the second-harmonic EPR spectra of the small spin probe di-tert-butyl nitroxide (DTBN) in these aggregates together with viscosity, conductivity, and DLS showed that, at pH ~ 7.54, MTNa formed micelles and MPNa vesicles and MTNa exhibited a pH-induced micelle to vesicle transition as pH was lowered toward 6. MTNa, at pH ~ 7.54, formed small micelles at low concentrations that transformed to long worm-like micelles for concentrations ≥ 0.05 M, accompanied by a 30-fold increase in solution viscosity. The hydrodynamic radii from DLS confirmed the presence of small micellar aggregates of radius ~ 2 nm in MTNa at pH ~ 7.54 at the lower concentrations, with coexisting micelles (~2 nm) and vesicles (~50 nm) at pH near 6.5, vesicles (radii ~ 70 nm) at pH near 6, and large vesicles (85 nm) in MPNa at pH ~ 7.60. Both MTNa and MPNa precipitated upon reduction of pH below 6 and below 7, respectively. The rate of transfer of DTBN between the aqueous phase and the aggregate was calculated from the high-field Lorentzian linewidths of the electron paramagnetic resonance (EPR) spectra. The activation energy for the transfer determined from the temperature dependence of the rate of transfer is 12.7 kJ/mol for MTNa vesicles (pH ~ 6) and 20.6 ± 1.3 kJ/mol for MPNa (pH ~ 7.60). The pH-induced transformations were reversible.

Partitioning of lysolipids, fatty acids and their mixtures in aqueous lipid bilayers: solute concentration/composition effects.

Distributions of lysopalmitoylphosphatidylcholine (LPPC), palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10% solute mole fraction than for 0 to 2%, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2% solute mole fraction. (2) Partition coefficients are in the order LPPC<PA<LPPC+PA at 37°C and LPPC+PA≤LPPC<PA at 50°C. This signifies synergism toward increased solute solubility in the bilayer-gel phase and lack of it in the bilayer-liquid phase when LPPC and PA are present together. Implications of the solute concentration/composition and bilayer phase dependences of the partition coefficients to the reported solute induced enhancements in transmembrane permeability are discussed.

Electronic states in GaAs/Al0.3Ga0.7As non-periodic superlattices

Transmission and photoluminescence measurements in GaAs/Al0.3As0.7As superlattices in which the layer widths vary randomly, illustrate the effects of one-dimensional non-periodic potentials on electronic energy states. The fundamental interband transition energy in the non-periodic structures is lower than that in the periodic case. A relation between this shift and the wavefunction localization length is derived. The PL emission from the non-periodic samples is ten to twenty times stronger than from the periodic samples.

Enhanced radiative decay in disordered GaAs/Al0.3Ga0.7As superlattices

Characteristics of the photoluminescence (PL) from disordered and ordered GaAs/Al0.3Ga0.7As superlattices are compared. The disordered superlattices (DSL) are nonperiodic and the ordered superlattices are periodic. The PL emission is much stronger in the DSLs by a factor that depends on the PL excitation wavelength and excitation power. These observations are interpreted in terms of an enhanced radiative decay rate in the DSLs brought about by the breakdown of translational symmetry in these systems.