Comparative studies on a mesophilic and a thermophilic α-amylase

A comparative study was performed on thermal stability of mesophilic and thermophilic α-amylases, and the effects of various denaturing agents, organic solvents, and stabilizers were investigated. As expected, the thermophilic enzyme showed higher resistance toward denaturation in water as its natural medium, but such a difference could not be detected in nonaqueous environments. Furthermore, stability of these molecules was improved by including various stabilizing agents. Of the compounds tested, sorbitol provided the highest degree of protection, which was found to be owing to its effect on increasing T _m and its ability in totally preventing deamidation of amino acid residues in the protein molecules.     

Synthesis of Some Novel 2‐Anilinothiophene, 2,3′‐Bithienyl and Thienylthiopyridine Derivatives Resistance to Penicillium Digitatum Effect the Fruit

Reaction of benzotriazol‐1‐yl acetone 1 with phenyl isothiocyanate followed with α‐chloroacetone or ethyl‐α‐chloroacetate afforded 2‐anilinothiophenes 3 or 4 , respectively. Treatment of 3 with malononitrile at different reaction conditions afforded 6 or 7 . Reaction of 1 with CS₂ in DMF and phenacylbromide afforded S‐alkylated thiophene 10 . Reactions of the latter compound with different active methylene nitriles afforded thienylthiopyridine derivatives 14 and 15 . Condensation of 10 with hydrazine hydrate afforded hydrazon derivative 16 . Reaction of thiophene 17 with formamide in DMF afforded 19 which converted to N‐thienylpyrimidine 20 when treated with malononitrile. The structure of the newly synthesized compounds has been established on the basis of their analytical and spectral data. The compounds were also investigated for antibacterial and antifungal activities.     

Adsorptive immobilization of submitochondrial particles on concanavalin a sepharose-4b

Submitochondrial particles (SMPs) prepared from beef liver mitochondria were immobilized on concanavalin A Sepharose-4B (Con A Sepharose). The process of immobilization was optimized by choosing an appropriate buffer system containing Mn²⁺ and Ca²⁺. Adsorption of SMPs on Con A-Sepharose was found to be a reversible process, nonelectrostatic in nature, and responsive to the presence of methyl α-d-glucopyranose and α-d-mannose. The involvement of membrane glycoproteins in the adsorption process was thus demonstrated. Further analysis of the data obtained on competition of binding by sugar molecules provided competition constants reflecting the potency of inhibition by each individual carbohydrate. Positive-cooperative interactions for binding to the matrix were observed especially at high concentrations of SMPs. The immobilized preparations were used successfully in continuous catalytic transformations involving succinate-cytochrome c reductase (SCR) an enzyme complex of the inner-mitochondrial membrane. Best results were obtained when such operations were carried out at 37?C.     

Molecular structure of an alkyl-side-chain polymer-water interface: origins of contact angle hysteresis

A new and direct approach to verify surface heterogeneity as the microscopic origin of contact-angle hysteresis is demonstrated. IR-visible sum-frequency-generation spectroscopy (SFG) was used to selectively probe the molecules at the interface of an alkyl-side-chain polymer [poly(vinyl n-octadecyl carbamate-co-vinyl acetate)] with water. The spectra indicate that in contact with water, the polymer surface is heterogeneous (having areas of differing surface energies). This evidence of surface heterogeneity supports the hysteresis observed in the advancing and receding contact angles of the polymer surface with water. The same measurements made for the chemically and structurally similar surface of an octadecyltrichlorosilane self-assembled monolayer indicates a homogeneous surface at the water interface. In this case, contact-angle hysteresis measurements implicate surface roughness as the cause of hysteresis. Atomic force microscopy measurements of roughness for these surfaces further support our conclusions. The polymer-water interface was probed using SFG at above-ambient temperatures, and an order-to-disorder transition (ODT) of alkyl side chains at the interface was observed, which closely follows the melting of crystalline side chains in the bulk. This transition explains the increased wettability of the polymer, by water, when the temperature is raised above the bulk melting temperature. Furthermore, the irreversibility of this ODT suggests that the disordered polymer-water interface is the thermodynamic equilibrium state, whereas the before-heating structure of this interface is a kinetically hindered metastable state.     

Spectrophotometric resolution of ternary mixtures of pseudoephedrine hydrochloride, dextromethorphan hydrobromide, and sodium benzoate in syrups using wavelength selection by net analyte signals calculated with hybrid linear analysis

Hybrid linear analysis (HLA), as a recent factor-based multivariate calibration technique, was applied for the spectrophotometric determination of ternary mixtures of pseudoephedrine hydrochloride (PSU), dextromethorphan hydrobromide (DXT), and sodium benzoate (BNZ). The utilized HLA was assisted by a wavelength selection procedure which was based on the calculation of the net analyte signal (NAS) regression plot in any considered wavelengths window for each test sample, in addition to a moving window strategy for searching the region with maximum linearity of NAS regression plot (minimum error indicator (EI)). HLA was applied because it was simpler to adapt to the NAS regression plot methodology, and also used less factors than partial least squares (PLS). An orthogonal array design was applied for formation of calibration and prediction sets in the concentration ranges 0-7500 μmol L⁻¹ for PSU, 0-300 μmol L⁻¹ for DXT, and 0-1400 μmol L⁻¹ for BNZ. The method had the ability to select wavelength regions that minimize the effect of non-linearity of the spectral data, in addition to that of non-modeled interferences. The application of the selected wavelength regions improved the obtained relative standard error of predictions for PSU, DXT, and BNZ, respectively, from 5.24, 8.67, and 5.48% to 2.19, 5.21, and 3.62% (using lower number of factors). To check the ability of the proposed method in selection of linear regions of spectra, a test for detecting non-linear regions of spectral data in multivariate spectroscopic assays was also described. Additives in the commercial syrup samples did not interfere with their determinations. The method was successfully applied for the determination of pseudoephedrine HCl, dextromethorphan HBr, and sodium benzoate in cough suppressant syrup samples.     

Infrared-visible sum frequency generation spectroscopic study of molecular orientation at polystyrene/comb-polymer interfaces

Surface-sensitive infrared-visible sum frequency generation spectroscopy (SFG) in total internal reflection geometry has been used to study the structure of poly(vinyl n-octadecyl carbamate-co-vinyl acetate) (PVNODC) or poly(octadecyl acrylate) (PA-18) in contact with a deuterated or hydrogenated polystyrene (dPS or hPS) layer. SFG spectra from the PVNODC (or PA-18)/hPS interface show methyl and methylene peaks corresponding to PVNODC (or PA-18) and phenyl peaks corresponding to the PS. Analysis suggests that the methyl groups are tilted at angles less than 30 degrees with respect to the surface normal. The presence of a strong methylene peak suggests the PVNODC alkyl side chains contain more gauche defects at the PS/PVNODC interface in comparison to PVNODC (or PA-18)/air interfaces. On heating, the SFG intensity from the PS/PA-18 interface drops sharply near the bulk melting temperature (T(m)) of PA-18. Interestingly, a similar drop in SFG signal is also observed for the PS phenyl groups. This demonstrates the ability of the phenyl group at the PS/PA-18 interface to rearrange itself upon the solid-to-liquid transition of the PA-18 alkyl side chain, at a temperature well below the bulk PS glass transition temperature. For PS/PVNODC interfaces, the drop in SFG intensity is gradual and in agreement with the three broad transitions of PVNODC observed in the bulk.     

Synthesis of 1,2,4-Triazol-3-ylmethyl-, 1,3,4-Oxa-, and -Thiadiazol-2-ylmethyl-1H-[1,2,3]-triazolo[4,5-d]pyrimidinediones

1-Carbethoxymethyl-4,6-dimethyl-1H-[1,2,3]triazolo[4,5-d]pyrimidine-5,7(4H,6H)-dione was synthesized and treated with hydrazine hydrate to give the corresponding hydrazide. The latter hydrazide was treated either with phenylisothiocyanate or with carbon disulfide/alc. KOH to afford the corresponding thiosemicarbazide and oxadiazole derivatives. Alkylation of 2-mercapto-1,3,4-oxadiazole with dimethyl sulfate or ethyl chloroacetate gave the corresponding 2-methylthio-, and 2-ethylthioglycolate derivatives. Formation of 1,3,4-thiadiazole, 5-mercapto-1,2,4-triazole, and 1,3,4-oxadiazole were carried out by treating of the latter thiosemicarbazide with conc. H₂SO₄, NaOH/HCl, and HgO. Treating of 5-mercapto-1,2,4-triazole with ethyl chloroacetate afforded the thioglycolate ester. Hydrolysis of the latter with hydrazine hydrate afforded the hydrazide derivatives. Condensation of these hydrazides with monosaccharide aldoses gave the corresponding sugar hydrazones. The novel compounds were tested for antiviral activity against hepatitis B virus and showed moderate activities.     

An accurate expression for radial distribution function of the Lennard-Jones fluid

A simple and accurate expression for radial distribution function (RDF) of the Lennard-Jones fluid is presented. The expression explicitly states the RDF as a continuous function of reduced interparticle distance, temperature, and density. It satisfies the limiting conditions of zero density and infinite distance imposed by statistical thermodynamics. The distance dependence of this expression is expressed by an equation which contains 11 adjustable parameters. These parameters are fitted to 353 RDF data, obtained by molecular dynamics calculations, and then expressed as functions of reduced distance, temperature and density. This expression, having a total of 65 constants, reproduces the RDF data with an average root-mean-squared deviation of 0.0152 for the range of state variables of 0.5  T^*  5.1 and 0.35  ρ^*  1.1 (T^*=kT/ε and ρ^* = ρσ³ are reduced temperature and density, respectively). The expression predicts the pressure and the internal energy of the Lennard-Jones fluid with an uncertainty that is comparable to that obtained directly from the molecular dynamics simulations.     

Role of axial ligand on the electronic structures of active intermediates in cytochrome P-450, peroxidase and catalase

DFT method (B3LYP) with 6-31G^* basis set was utilized in the computation of a fully optimized structure, net atomic charges and spin densities of the intermediate of cytochrome P-450-oxoiron(IV) porphyrin cation radical, compound I – in the presence of axial ligand such as thiolate (SMe⁻) imidazole (IM), phenoxide (OPh⁻), methoxide (OMe⁻) and chloride (Cl⁻). The results show doublet states in compound I are about 2–4 kcal/mol more stable than quartet states for all aforementioned ligands, and the doublet state is the ground state in all cases. However, electron donor ability of the ligands are in the order of SMe⁻ > IM > OMe⁻ > OPh⁻ > Cl⁻. Also the active oxidant intermediate of cytochrome P-450 between different mesomeric structures select sulfur oxygen radical type structure and can be viewed as (RS^↓)Fe^↑(IV)(O^↑)(Por). In horseraddish peroxidase (HRP) and peroxidase with histidine axial ligand π cation radical character of porphyrin ring is preferred (Im)Fe^↑(IV)(O^↑)(Por^↓). For the ligands such as OMe⁻, OPh⁻ and Cl⁻ oxidation mainly took place on the iron and the active intermediate can be viewed as (L)Fe^↑(V)(O)(Por) with one unpaired electron localized on the iron.     

Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis

The enantiomeric separation of some nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated in capillary electrophoresis (CE) using dual systems with mixtures of charged cyclodextrin (CD) derivatives. A significant enhancement of selectivity and resolution could be achieved in the enantioseparation of these analytes in their uncharged form by the simultaneous addition of two oppositely charged CD derivatives to the background electrolyte. The combination of the single-isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-beta-CD (PMMAbetaCD) and the single-isomer polyanionic CD, heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) in a pH 2.5 phosphoric acid-triethanolamine buffer, was designed and employed for the enantioseparation of profens. The improvement in selectivity and resolution can be attributed to the fact that the two CDs, which lead to independent and enantioselective complexation with the analyte enantiomers, have not only opposite effects on the electrophoretic mobility of these compounds but also opposite affinity patterns towards the enantiomers of these compounds. Binding constants for these enantiomers with each CD were determined using linear regression approach, in order to be able to predict the effect of the concentrations of the two CDs on enantiomeric selectivity and resolution in such dual systems.