Effects of organic solvents and substrate binding on trypsin in acetonitrile and hexane media

In this work, we used molecular dynamic (MD) simulation to study trypsin with and without a six-amino-acid peptide bound in three different solvents (water, acetonitrile and hexane) in order to provide molecular information for well understanding the structure and function of enzymes in non-aqueous media. The results show that the enzyme is more compact and less native-like in hexane than in the other two polar solvents. The substrate could stabilize the native protein structure in the two polar media, but not in the non-polar hexane. There are no significant differences in the conformation of the S1 pocket upon the substrate binding in water and acetonitrile media while a reverse behavior is observed in hexane media, implying a possible induced fit binding mechanism in the non-polar media. The substrate binding enhances the stability of catalytic H-bond network since it could expel the solvent molecules from the active site. The enzyme and the substrate appear to be more appropriate to the reactive conformation in the organic solvents compared with aqueous solution. There is much greater substrate binding strength in hexane media than the water and acetonitrile ones since the polar solvent significantly weakens electrostatic interactions, which are observed to be the main driving force to the binding. In addition, some residues of the S1 pocket could remain favorable contribution to the binding despite the solvent change, but with differences in the contribution extent, the number and the type of residues between the three media.

Simultaneous determination of nine fluoroquinolones in egg white and egg yolk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method with fluorescence detection was developed for determination of nine fluoroquinolones (FQs) in egg white and yolk. Egg white samples were deproteinized with acidified ethanol (egg yolk samples with acetonitrile and acidified ethanol), followed by defatting with hexane once (white) or twice (yolk), and extracting FQs into acetonitrile. After acetonitrile was evaporated, the residue was dissolved in mobile phase, and FQs were detected in LC with a fluorescence detector. Recoveries for nine FQs from white and yolk were 74.7-85.6%, 79.1-91.2%, respectively, with excellent relative standard deviations. The limits of quantification were 5-20 ngg(-1).     

Solvent extraction of tricyclic amines from blood plas- ma and liquid chromatographic determination

The extraction of seven tricyclic antidepressant amines from human plasma at different pH values was investigated for dichloromethane, diethyl ether and hexane—1-pentanol (95:5). The amines were extracted as bases and back-extracted to sulphuric acid, 0.10 mol/l, prior to the separation by bonded-phase liquid chromatography. Ether and hexane— 1-pentanol (95:5) were most suitable, tertiary amines being best extracted at pH 8, and secondary amines at pH 10. Using ether, both tertiary and secondary amines required 30 min extraction time for a quantitative yield while 15 rain was sufficient for hexane— -1-pentanol (95:5). UV detection allowed concentrations down to 10 ng in 1 ml of plasma to be determined.Three ammonium ions—octylammonium, dimethylammonium, and trimethylammonium - were added as modifiers to the mobile phase containing acetonitrile in phosphoric acid, 0.10 mol/l. In the concentration interval 0.010–0.030 mol/l all of the amine modifiers gave on Polygosil C₈ peak asymmetry factors of sufficiently low magnitude, while on Li- Chrosorb RP-18 this was so only for di- and trimethylammonium in a concentration of 0.030 mol/l.     

Bioreduction of a carbon–nitrogen double bond using immobilized baker's yeast’a first report

Immobilized baker's yeast entrapped in calcium alginate beads efficiently reduces N-benzylidinemethylamine to N-methylbenzylamine in hexane at 37°C and tetrahydrofuran (THF) at 30°C in the presence of 18-crown-6, while in the presence of water as cosolvent and glucose as an additive N-benzylidinemethylamine undergoes decomposition. Benzaldoxime in a hexane–water (1:9) solvent system containing glucose as an additive is reduced to N-benzylhydroxylamine. On using an ethanol–water (1:1) solvent system, benzaldoxime is converted to benzyl alcohol and in hexane, benzene, THF, hexane–water (1:1) or acetonitrile–water (1:1) solvent systems, or using dried baker's yeast in different solvent systems, transformation of benzaldoxime does not occur.     

Effects of water content on the tetrahedral intermediate of chymotrypsin - trifluoromethylketone in polar and non-polar media: observations from molecular dynamics simulation

The work uses MD simulation to study effects of five water contents (3 %, 10 %, 20 %, 50 %, 100 %?v/v) on the tetrahedral intermediate of chymotrypsin - trifluoromethyl ketone in polar acetonitrile and non-polar hexane media. The water content induced changes in the structure of the intermediate, solvent distribution and H-bonding are analyzed in the two organic media. Our results show that the changes in overall structure of the protein almost display a clear correlation with the water content in hexane media while to some extent U-shaped/bell-shaped dependence on the water content is observed in acetonitrile media with a minimum/maximum at 10–20 % water content. In contrast, the water content change in the two organic solvents does not play an observable role in the stability of catalytic hydrogen-bond network, which still exhibits high stability in all hydration levels, different from observations on the free enzyme system [Zhu L, Yang W, Meng YY, Xiao X, Guo Y, Pu X, Li M (2012) J Phys Chem B 116(10):3292–3304]. In low hydration levels, most water molecules mainly distribute near the protein surface and an increase in the water content could not fully exclude the organic solvent from the protein surface. However, the acetonitrile solvent displays a stronger ability to strip off water molecules from the protein than the hexane. In a summary, the difference in the calculated properties between the two organic solvents is almost significant in low water content (<10 %) and become to be small with increasing water content. In addition, some structural properties at 10?~?20 %?v/v hydration zone, to large extent, approach to those in aqueous solution.

Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons

The protonation of crotonyl-CoA dienolate following decarboxylation of glutaconyl-CoA by glutaryl-CoA dehydrogenase was investigated. Although it is generally held that the active sites of acyl-CoA dehydrogenases are desolvated when substrate binds, recent evidence has established that water has access to the active site in these binary complexes of glutaryl-CoA dehydrogenase. The present investigation shows that the dehydrogenase catalyzes (a) a rapid exchange of C-4 methyl protons of crotonyl-CoA with bulk solvent and (b) protonation of crotonyl-CoA dienolate by solvent-derived protons under single turnover conditions. Both of the reactions require the catalytic base, Glu370. These findings indicate that decarboxylation proceeds via a dienolate intermediate. The involvement of water in catalysis by glutaryl-CoA dehydrogenase was previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.     

Asymmetric bioreduction of keto esters by immobilized baker's yeast entrapped in calcium alginate beads in both water and organic/water solvent systems

Immobilized baker's yeast entrapped in calcium alginate beads, ca 1.5 mm diameter, was used more than 10 times in water and reduced ethyl 3-oxobutanoate and ethyl benzoylformate to the corresponding chiral hydroxy esters in good chemical yields and in high enantioselectivities. The biocatalyst was also successfully used in organic/water solvent systems such as hexane/water and acetonitrile/water, and in other systems, particularly in hexane, converted keto esters into their individual chiral hydroxy esters.     

Characterisation of the lowest singlet and triplet excited states of S-flurbiprofen.

The photophysical properties of S-flurbiprofen [S-2-fluoro-alpha-methyl-4-biphenylacetic acid], a nonsteroidal anti-inflammatory drug, have been examined using steady-state and time-resolved spectroscopic techniques. The energy of its first singlet excited state is 99 kcal mol(-1). The fluorescence quantum yields and lifetimes (at 300 nm) have been determined in acetonitrile, methanol, hexane and PBS; they are in the range 0.15相似文献   

Fluorescence Detection of Free Radicals by Nitroxide Scavenging

The fluorescence quantum yield of 4-(1-napthoyloxy)-2,2,6,6-tetramethylpiperidine-l-oxyl (I) in acetonitrile and hexane is 55 and 30-fold lower, respectively, than those of diamagnetic analogs. Experiments described herein demonstrate that this property makes possible the fluorescence detection of radical scavenging reactions in which the paramagnetic nitroxide-substituted naphthalene is converted to a diamagnetic N-alkoxy derivative. 2-Cyanopropyl free radicals were generated by the thermal decomposition of azobisisobutyronitrile (AIBN) in cyclohexane or in acetonitrile containing 1. The fluorescence intensity of the sample increased proportionally to the decrease in its ESR signal intensity, indicating the conversion of the paramagnetic nitroxide to the diamagnetic product. The linear relationship between the increase in fluorescence intensity and decrease in ESR signal intensity shows that the changes in the fluorescence intensity can serve as a sensitive means for optically detecting radicals.     

Fluorescence Detection of Free Radicals by Nitroxide Scavenging

The fluorescence quantum yield of 4-(1-napthoyloxy)-2,2,6,6-tetramethylpiperidine-l-oxyl (I) in acetonitrile and hexane is 55 and 30-fold lower, respectively, than those of diamagnetic analogs. Experiments described herein demonstrate that this property makes possible the fluorescence detection of radical scavenging reactions in which the paramagnetic nitroxide-substituted naphthalene is converted to a diamagnetic N-alkoxy derivative. 2-Cyanopropyl free radicals were generated by the thermal decomposition of azobisisobutyronitrile (AIBN) in cyclohexane or in acetonitrile containing 1. The fluorescence intensity of the sample increased proportionally to the decrease in its ESR signal intensity, indicating the conversion of the paramagnetic nitroxide to the diamagnetic product. The linear relationship between the increase in fluorescence intensity and decrease in ESR signal intensity shows that the changes in the fluorescence intensity can serve as a sensitive means for optically detecting radicals.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Measuring corticosterone in feathers using an acetonitrile/hexane extraction and enzyme immunoassay: feather corticosterone levels of food‐supplemented Atlantic Puffin chicks

Glucocorticoid levels measured in the blood of animals reflect hypothalamic‐pituitary‐adrenal (HPA) activity in response to predictable and unpredictable changes. In birds, circulating corticosterone is incorporated into growing feathers and provides an integrated measure of HPA activity over the period of feather growth. Measuring corticosterone in feathers can provide insight into the physiological state of birds during times when they are unavailable for blood sampling (e.g., during migratory or non‐breeding periods of the annual cycle). Building upon studies that used radioimmunoassay or liquid chromatography, we used a commercially available enzyme immunoassay kit to measure corticosterone in feathers of nestling Atlantic Puffins (Fratercula arctica) on Gull Island, Newfoundland, Canada, in 2012, and demonstrate the benefits of sample preparation via acetonitrile/hexane purification. We used this method to measure corticosterone in feathers of Atlantic Puffin chicks that experienced differences in mass gain in a supplementary feeding study. We found a positive relationship between feather corticosterone and mass gain, and a negative relationship between feather corticosterone and pre‐treatment body condition. Because feathers were growing prior to and during the supplementary feeding period, our results also suggest that extracting seabird feather samples with acetonitrile/hexane (in addition to methanol) prior to measuring corticosterone with enzyme immunoassay is beneficial, and, as reported in previous studies, blood and feather corticosterone values reflect different measures.     

Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.     

GC-MS analysis of compounds extracted from buds of Populus balsamifera and Populus nigra

The composition of hexane and ether extracts from buds of two poplar species (Populus balsamifera and P. nigra) was investigated by GC-MS method. In hexane extracts, 54 "neutral" compounds were recorded. The greatest amounts of them are sesquiterpenes and n-alkanes. Among 56 components of ether extracts, many aliphatic acids and hydroxyacids were detected. However, the main fraction consists of phenolcarboxylic acids, substituted cinnamic acids, and their esters. It was established that chemotaxonomic differences between Populus balsamifera and P. nigra are observed in the case of both hexane and ether bud extracts.     

Fluorescence derivatisation of urinary corticosteroids for high-performance liquid chromatographic analysis

Corticosteroids containing a C₂₁ primary hydroxyl group were derivatised with 9-anthroyl cyanide. The reagent was prepared as a solution in acetonitrile, containing 0.1% triethylamine, at a concentration of 2 mg/ml. Approximately 1 μg of corticosteroid was reacted with 100 μl of this reagent, at 45°C for 2 h. The fluorescent derivatives were separated by HPLC on a silica column, 250×4.6 mm I.D., by stepwise elution, with a mobile phase of 2-propanol–hexane (2:98) for 20 min, followed by 2-propanol–hexane (7:93) from 20 to 40 min. The fluorescence detector was set to 370-nm excitation and 470-nm emission. The relatively low temperature for derivatisation avoided reaction with secondary hydroxyl groups and also prevented thermal degradation of the corticosteroids.     

Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions

The purpose of this study was to determine the effect of water and hexane soluble fractions of the Indian medicinal plant, Solanum trilobatum on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish were intraperitoneally injected with different doses of 0, 4, 40 or 400 mg kg(-1) body weight of water or hexane soluble fraction. The nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, reactive oxygen species production and reactive nitrogen species production by peripheral blood leucocytes. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) was assessed by a challenge with live Aeromonas hydrophila. Almost all the doses of both water and hexane soluble fractions enhanced the serum lysozyme activity. All the doses of water soluble fraction significantly enhanced the ROS production on most of the days tested. In hexane soluble fraction treated groups, the enhancement in the ROS production was observed at least on 2 days. All the doses of water soluble fraction significantly enhanced the production of RNS only on one day. The RNS production was enhanced significantly only in the group treated with 40 mg kg(-1) of hexane fraction. The leaf fraction administration preceding the challenge with live A. hydrophila, decreased the percentage mortality in the experimental group with the consequent increase in RPS values. This preliminary study indicates that S. trilobatum could be used to promote the health status of fish in intensive finfish aquaculture.     

Effect of culture conditions at lab-scale on metabolite composition and antibacterial and antibiofilm activities of Dunaliella tertiolecta

Dunaliella tertiolecta RCC6 was cultivated indoors in glass bubble column photobioreactors operated under batch and semi-continuous regimens and using two different conditions of light and temperature. Biomass was harvested by centrifugation, frozen, and then lyophilized. The soluble material was obtained by sequential extraction of the lyophilized biomass with solvents with a gradient of polarity (hexane, ethyl acetate, and methanol) and its metabolic composition was investigated through nuclear magnetic resonance (NMR) spectroscopy. The effect of light on chlorophyll biosynthesis was clearly shown through the relative intensities of the ¹H NMR signals due to pheophytins. The highest signal intensity was observed for the biomasses obtained at lower light intensity, resulting in a lower light availability per cell. Under high temperature and light conditions, the ¹H NMR spectra of the hexane extracts showed an incipient accumulation of triacylglycerols. In these conditions and under semi-continuous regimen, an enhancement of β-carotene and sterols production was observed. The antibacterial and antibiofilm activities of the extracts were also tested. Antibacterial activity was not detected, regardless of culture conditions. In contrast, the minimal biofilm inhibitory concentrations (MBICs) against Escherichia coli for the hexane extract obtained under semi-continuous regimen using high temperature and irradiance conditions was promising.     

Antioxidant activities of probucol against lipid peroxidations.

The antioxidant activities of probucol were measured in the oxidations of methyl linoleate in homogeneous solution and soybean phosphatidylcholine liposomal membranes and also of low-density lipoproteins. When an excess amount of probucol was reacted with galvinoxyl, the EPR spectrum of galvinoxyl disappeared and a new triplet EPR signal was found: g = 2.0058 and aH(2H) = 0.14 mT. The identical EPR spectrum was observed when probucol was reacted with tert-butoxyl radical generated from di-tert-butylperoxy oxalate. This EPR signal disappeared rapidly when reacted with either alpha-tocopherol or 6-O-palmitoyl-ascorbic acid. Probucol suppressed the free-radical-mediated oxidations of methyl linoleate in hexane and in acetonitrile, in a dose-dependent manner. Its antioxidant activity was 17.5-fold less than that of alpha-tocopherol in hexane. Probucol incorporated into soybean phosphatidylcholine liposomes suppressed its oxidation. The antioxidant activity of probucol was less than that of alpha-tocopherol, but the difference between the two antioxidant activities was smaller in the membranes than in homogeneous solution. Probucol also suppressed the oxidation of low-density lipoprotein. Interestingly, probucol suppressed the oxidation of LDL as efficiently as alpha-tocopherol, implying that physical factors as well as chemical reactivity are important in determining the overall activity of antioxidant in low-density lipoprotein.