Mahinda Gangoda

Ionization properties of phosphatidylinositol polyphosphates in mixed model membranes.

Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P(2) in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P(2) is clustered even at 1 mol %.

Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

TEABr directed synthesis of ZSM-12 and its NMR characterization

Abstract Synthesis of ZSM-12 using tetraethylammonium bromide (TEABr) as the template was investigated. Among the various parameters that affect the crystallization of ZSM-12, aluminum content of the gel, OH − /SiO 2 and TEA/SiO 2 ratios were the important determinants. Systematic variations of these parameters revealed that the TEABr-assisted synthesis had many similarities to the synthesis using TEAOH template reported earlier, with the exception that the OH − /SiO 2 ratios had to be maintained at lower values. Furthermore, the OH − /SiO 2 ratios favorable for ZSM-12 formation lie in a very narrow range. The source of alkalinity also affected the rate of crystallization and the composition of the product. The crystallization was found to be faster and better incorporation of aluminum in the zeolite framework was obtained when NaOH was used to provide alkalinity rather than KOH. Successful synthesis of highly crystalline ZSM-12 samples with Si/Al ratio around 30 was achieved using a minimal amount of relatively inexpensive TEABr (TEA/SiO 2 =0.125). Aluminum-27 NMR spectroscopy unambiguously revealed that all aluminum atoms are incorporated in the zeolite framework in tetrahedral coordination. The NMR line-widths of aluminum signals of the calcined samples were significantly larger than those with template incorporated samples. Spin-lattice relaxation times, conventional and rotating frame, as well as magic angle spinning (MAS) cross-polarization data with variable contact time support that there is a significant proton reservoir in the aluminum framework. The NMR data indicate that many distorted tetrahedral sites are formed upon removal of the template and some of these sites contain Al–OH moieties.

Chromatographic and related studies of alkylamide phases

SummaryA series of chemically bonded phases has been prepared with amide groups localized by means of hydrophobic ligands. The physicochemical and chromatographic properties of such phases with chain lengths ranging from C5 to C8 have been examined by porosimetry, elemental analysis, solid-state NMR, and liquid chromatography. Subsequently, the conformational dynamics of these phases have been investigated by analyzing the dependence of the capacity factor (k) on the reciprocal of temperature for different organic compounds. Special emphasis has been given to the reproducibility of retention data obtained before and after temperature-dependent measurements.

Characterization of silica-based octyl phases of different bonding density: Part II. Studies of surface properties and chromatographic selectivity

The goal of the current work was to evaluate changes in the surface composition of the stationary phase as a function of the bonded ligand density. To fulfill this purpose several octyl phases of different surface coverage were synthesized from the same support silica. For each chromatographic system the methylene selectivity measurements were used to estimate the equilibrium sorption constant and surface excess of acetonitrile in the stationary phase. The differences in the stationary phase composition were correlated with the surface properties of the corresponding chromatographic packings, which were characterized by nitrogen adsorption, thermogravimetry and solid-state NMR.

Reversed-phase liquid chromatographic studies of non-ionic surfactants and related solutes

Abstract Homologous series of alkyl ester, ether and amide polyoxyethylene non-ionic surfactants have been synthesized and their retention properties have been studied under reversed-phase conditions. In addition, methylene group selectivities have been determined for the three types of surfactants as well as for a homologous series of ethyl esters of alkanoic acids. In the composition range studied for a given mobile phase, methylene selectivity was nearly independent of the head group of the surfactants. This supports the idea that the retention mechanism is governed by hydrophobic interactions between the alkyl tail of the surfactant and the bonded stationary phase.

Preparation and characterization of silica-carbon hybrids

Abstract Silica-carbon (SC) hybrids of varying composition have been prepared by a cyclic two-step process which consisted of: (1) initially adsorbing silicon tetrachloride onto the surface of porous carbon particles, and (2) converting silicon tetrachloride into silica via reaction with water vapor. At the completion of each cycle all samples were studied by thermogravimetric analysis and 29 Si CP-MAS NMR spectrometry in order to determine the amount of deposited silica and to characterize their surface and structural properties. Further, the adsorption capacities of the SC hybrids were measured chromatographically.

Mesoporous isocyanurate-containing organosilica–alumina composites and their thermal treatment in nitrogen for carbon dioxide sorption at elevated temperatures

Composite mesostructures consisting of organosilica with isocyanurate bridging groups and alumina have been synthesized using evaporation-induced self-assembly (EISA) in the presence of a triblock copolymer, Pluronic P123, in absolute ethanol solution. These mesostructures were prepared using aluminum isopropoxide and aluminum nitrate nonahydrate as alumina precursors and tris[3-(trimethoxysilyl)propyl]isocyanurate (ICS). The triblock copolymer was removed by extraction with 95% ethanol solution followed by additional thermal treatment of the extracted sample at 300 °C in flowing nitrogen; this process assured a complete removal of the polymeric template without degradation of the ICS bridging groups. The use of aluminum nitrate nonahydrate and a N-containing ICS precursor with a small amount of 3-aminopropyltriethoxysilane (AP) led to the hybrid materials with well-developed porosity and high specific surface area (200–450 m2 g−1). A controlled heating of these materials in nitrogen resulted in N-doped alumina–silica mesostructures showing high affinity towards CO2 at elevated temperatures. The use of inexpensive aluminum nitrate instead of aluminum alkoxides in the EISA synthesis had a significant impact on the pore structure, surface area and adsorption properties of the resulting composite materials.

Characterization and multi-mode liquid chromatographic application of 4-propylaminomethyl benzoic acid bonded silica—A zwitterionic stationary phase

4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by (13)C cross polarization magic angle spinning ((13)C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, L-arginine, L-phenylalanine, and L-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation.

Comparative study of alkyl and fluoroalkyl N-(2-hydroxy-ethoxyethyl)-amides as reversed-phase liquid chromatographic modifiers

Abstract The use of the non-ionic surfactant, N-(2-hydroxy-ethoxyethyl)-2,2,3,3,4,4,4-heptafluorbutanamide as a liquid chromatographic mobile phase modifier has been studied. Comparisons between this fluorinated compound and two similar hydrocarbon surfactants, N-(2-hydroxyethoxyethyl)-hexanamide and N-(2-hydroxyethoxyethyl)-heptanamide, have been made. Although surface tension data were similar for all three surfactants, the fluoroalkyl compound was found to have a larger influence on retention. Likewise, the positional isomers of cresol and toluidine were resolvable using the fluorinated surfactant and were not with the equivalent alkyl surfactant with a similar hydrophilic-lipophilic balance.