Product-retardation and -activation of catalytic hydrolysis by phospholipase D in small unilamellar vesicles of egg yolk phosphatidylcholine

 We evaluated the hydrolysis of egg yolk phosphatidylcholine (PC) by phospholipase D from Streptomyces chromofuscus (PLD) in small unilamellar vesicles (SUV) in presence of 50 μM Ca²⁺. After initial choline production (hydrolysis of 1.5% of the PC at the outer leaflets of the vesicle bilayers), the hydrolysis was reduced to 5% of the initial velocity. The kinetic behavior in SUV of premixed PC and a low percentage of the hydrolysis product, phosphatidic acid (PA), was similar to that of PC SUV. The reduced velocity disappeared when the membrane structure was disintegrated by means of a nonionic surfactant. In the retardation phase, the partially hydrolyzed vesicles (postsubstrates) had much higher affinity for PLD than fresh PC SUV. These results indicated that small clusters of the product, PA, at the vesicle surface were responsible for the reduced velocity of hydrolysis. The initial velocity increased in a biphasic manner with the substrate concentration. At a PC concentration range up to 4 mM, the experimental data fit Michaelis–Menten kinetics. At concentrations above 6 mM, the velocity again markedly increased. Negatively charged mixed vesicles of PC and PA did not have such kinetics. Furthermore, adding PC SUV to the postsubstrates, where the fraction of free PLD was less than 0.05, induced steep choline production. These results showed that PLD bound to vesicles had higher activity than free PLD. We speculated that PLD bound to vesicles collided with and was directly transferred to PC SUV when the fraction of free PLD in aqueous medium was very small. Received: 5 November 1996 Accepted: 26 February 1997     

A study of macroderm a and macroderm L monolayers and their two-dimensional compatibility

 Spread monolayers of two new skin permeation enhancers, MacroDerm A and MacroDerm L were investigated at the water/air interface as a function of temperature and of subphase composition. Both components did not seem to be markedly affected by changes in ionic strength and by the presence of metal ions in the subphase. The two-dimensional binary system MacroDerm A –MacroDerm L was also studied at the water/air interface at 298 K on pure water subphase. The behavior of surface areas, surface compressional moduli and collapse pressure as a function of molar ratios of components shows that MacroDerm A and MacroDerm L are miscible. Received: 17 December 1996 Accepted: 5 March 1997     

Relationships between equilibrium spreading pressure and phase equilibria of phospholipid bilayers and monolayers at the air-water interface

The intricate interplay between the bilayer and monolayer properties of phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) phospholipids, in relation to their polar headgroup properties, and the effects of chain permutations on those polar headgroup properties have been demonstrated for the first time with a set of time-independent bilayer-monolayer equilibria studies. Bilayer and monolayer phase behavior for PE is quite different than that observed for PC and PG. This difference is attributed to the characteristic biophysical PE polar headgroup property of favorable intermolecular hydrogen-bonding and electrostatic interactions in both the bilayer and monolayer states. This characteristic hydrogen-bonding ability of the PE polar headgroup is reflected in the condensed nature of PE monolayers and a decrease in equilibrium monolayer collapse pressure at temperatures below the monolayer critical temperature, T(c) (whether above or below the monolayer triple point temperature, T(t)). This interesting phenomena is compared to equilibrated PC and PG monolayers which collapse to form bilayers at 45 mN/m at temperatures both above and below monolayer T(c). Additionally, it has been demonstrated by measurements of the equilibrium spreading pressure, pie, that at temperatures above the bilayer main gel-to-liquid-crystalline phase-transition temperature, T(m), all liquid-crystalline phospholipid bilayers spread to form monolayers with pie around 45 mN/m, and spread liquid-expanded equilibrated monolayers collapse at 45 mN/m to form their respective thermodynamically stable liquid-crystalline bilayers. At temperatures below bilayer T(m), PC and PG gel bilayers exhibit a drop in bilayer pi(e) values < or =0.2 mN/m forming gaseous monolayers, whereas the value of pic of spread monolayers remains around 45 mN/m. This suggests that spread equilibrated PC and PG monolayers collapse to a metastable liquid-crystalline bilayer structure at temperatures below bilayer T(m) (where the thermodynamically stable bilayer liquid-crystalline phase does not exist) and with a surface pressure of 45 mN/m, a surface chemical property characteristically observed at temperatures above bilayer T(m) (monolayer T(c)). In contrast, PE gel bilayers, which exist at temperatures below bilayer T(m) but above bilayer T(s) (bilayer crystal-to-gel phase-transition temperature), exhibit gel bilayer spreading to form equilibrated monolayers with intermediate pie values in the range of 30-40 mN/m; however, bilayer pie and monolayer pic values remain equal in value to one another. Contrastingly, at temperatures below bilayer T(s), PE crystalline bilayers exhibit bilayer pie values < or =0.2 mN/m forming equilibrated gaseous monolayers, whereas spread monolayers collapse at a value of pic remaining around 30 mN/m, indicative of metastable gel bilayer formation.     

Micron-sized monodispersed polymer particles produced by seeded polymerization for the dispersion of high swelling of polymer particles with a large amount of monomer

 Micron-sized monodispersed polystyrene (PS)/poly(n-butyl methacrylate) composite particles were produced as follows. First, 1.77 μm-sized monodispersed PS seed particles produced by dispersion polymerization were dispersed in ethanol/water (1/2, w/w) medium dissolving poly(vinyl alcohol) as a stabilizer. n-Butyl methacrylate (BMA) monomer dissolving benzoyl peroxide initiator was emulsified in ethanol/water (1/2, w/w) solution of sodium dodecyl sulfate as emulsifier with ultrasonic homogenizer, and the BMA monomer emulsion was mixed with the PS seed emulsion. The PS seed particles absorbed with a large amount of BMA (about 150 times weight of the seed particles) for 2 h to about 10 μm in diameter while keeping good monodispersity and BMA droplets disappeared finally. The seeded polymerization was carried out at 70 °C after a certain amount of water was added to depress the redissolving of BMA from the swollen particles into the medium by raising from room temperature to the polymerzation temperature. Received: 21 February 1996 Accepted: 4 September 1996     

Determination of copper by anodic stripping voltammetry on a glassy carbon electrode using a continuous flow system

 A successful flow-through system was developed for trace analysis of copper using DPASV with a glassy-carbon electrode. Periodical chemical regeneration of the electrode with a 1 mol/L NaOH solution increased sensitivity and precision. The method was shown to be applicable with a detection limit of 0.56 μg/L, with a determination time of less than 7 min per measurement (without deaeration time). The drawback of the system is the 10 min deaeration time. The system gave an accuracy of 0.090±0.005% for a certified reference material of low alloy steel containing 0.090±0.004% Cu. Applicability to various fresh water samples with a Cu content between 1.57 and 13.11 μg/L with an RSD<2.36% is illustrated. Received: 11 March 1996/Revised: 1 July 1996/Accepted: 4 July 1996     

Determination of copper by anodic stripping voltammetry on a glassy carbon electrode using a continuous flow system

 A successful flow-through system was developed for trace analysis of copper using DPASV with a glassy-carbon electrode. Periodical chemical regeneration of the electrode with a 1 mol/L NaOH solution increased sensitivity and precision. The method was shown to be applicable with a detection limit of 0.56 μg/L, with a determination time of less than 7 min per measurement (without deaeration time). The drawback of the system is the 10 min deaeration time. The system gave an accuracy of 0.090±0.005% for a certified reference material of low alloy steel containing 0.090±0.004% Cu. Applicability to various fresh water samples with a Cu content between 1.57 and 13.11 μg/L with an RSD<2.36% is illustrated. Received: 11 March 1996/Revised: 1 July 1996/Accepted: 4 July 1996     

Enhanced thermal stability and structural ordering in short chain <Emphasis Type="Italic">n</Emphasis>-alkanethiol monolayers on gold probed by vibrational spectroscopy and EQCM

Monolayers of alkanethiols with varied chain lengths, CH₃(CH₂)_nSH where n = 3, 5, and 7, on gold substrates have been prepared by adsorption from (1) neat thiol, (2) millimolar thiol solution in alcohol (conventional method), and (3) potential-controlled adsorption. Reflection absorption infrared spectroscopy (RAIRS) and electrochemical quartz crystal microbalance (EQCM) have been used to characterize the integrity of the monolayers. Methylene and methyl stretching modes along with C–S stretching modes have been used as benchmarks to follow the order–disorder transitions in the monolayer structure, in the temperature range from 25 to 175 °C. Monolayers adsorbed from neat thiol show superior quality in terms of stability and structural arrangement. Short chain thiols with n = 3, 5, and 7 do show substantial stability. The possibility of multilayer formation is ruled out by EQCM studies comparing the frequency and mass change associated with the monolayer desorption. Self-assembled monolayers (SAMs) adsorbed under potential control behave very similarly to the monolayers adsorbed from neat thiol as far as stability and structural orientation are concerned, irrespective of the chain length. The adsorption from neat thiol gets rid of the solvent-induced defects and arrests the propagation of defects under temperature constraints.     

Determination of palladium by adsorptive stripping voltammetry

 Adsorptive accumulation of the Pd(II) complex with dimethylglyoxime was evaluated for stripping voltammetry with respect to different parameters. The sensitivity of the method and the linearity between the peak current and the concentration of Pd(II) depends on the ionic strength, the electrode area, the preconcentration time, the transport rate to the electrode, and the potential scan rate. The most appropriate medium was 0.1 mol/L acetate buffer between pH 3.5 and 4. Using 2 min of preconcentration at a 2.6 mm² electrode and the differential pulse mode, a detection limit of 0.05 μg/L Pd was achieved for liquid samples and 50 ng/g for solid samples. Different aqueous and solid samples were analysed and the recovery from biological and inorganic materials investigated. Received: 29 February 1996/Revised: 29 July 1996/Accepted: 1 August 1996     

The magnitude of condensation induced by cholesterol on the mixtures of sphingomyelin with phosphatidylcholines-Study on ternary and quaternary systems

The studies on the condensing and ordering effect of cholesterol by application of the Langmuir monolayer technique are usually performed on binary lipid/cholesterol systems. The results concerning a quantitative analysis of these effects in multicomponent monolayers are very limited. In this work the condensing and ordering effect of cholesterol in ternary (SM/DSPC/Chol and SM/DOPC/Chol) and quaternary (SM/DSPC/DOPC/Chol) films was investigated. It was evidenced that the systems containing saturated PC (both SM/DSPC and SM/DSPC/Chol) are always more condensed and chain-ordered than the systems containing unsaturated PC (SM/DOPC and SM/DSPC/DOPC and their mixtures with cholesterol). However, the magnitude of condensation provoked by cholesterol at higher surface pressures is stronger on the monolayers containing unsaturated PC. The addition of cholesterol into SM/PC films induces the increase of chain-ordering however, the effectiveness of cholesterol as an ordering agent is determined by the presence/absence of unsaturated phospholipid. The magnitude of the effect of cholesterol on the investigated mixed monolayer was analyzed in the context of the influence of sterol on lipid chains (ordering, straightening and reorientation of chains) as well as the reorientation of polar heads.     

Spectrophotometric determination of nitrite in natural waters using diazotization-coupling method with column preconcentration on naphthalene supported with ion-pair of tetradecyldimethylbenzyl-ammonium and iodide

 A column preconcentration method has been established for the spectrophotometric determination of traces of nitrite using diazotization and coupling on an naphthalene-tetradecyldimethylbenzylammonium (TDBA)-iodide (I) adsorbent. Nitrite ion reacts with sulfanilic acid (SA) in the pH range 1.8–3.0 for the SA-1-naphthol system and in the pH range 2.3–3.2 for the SA-1-naphthylamine-4-sulfonate system (SA-NAS system) in hydrochloric acid medium to form water-soluble colourless diazonium cations. These cations were coupled with 1-naphthol in the pH range 1.6–4.6 and with NAS in the pH range 2.6–5.0 to be retained on naphthalene-TDBA-I packed in a column. The solid mass was dissolved from the column with 5 mL of dimethylformamide (DMF) and the absorbance measured at 418 nm for the SA-1-naphthol system and at 485 nm for the SA-NAS system. The calibration curve was linear over the concentration range 0.02–0.87 mg/L for SA-1-naphthol and 0.02–0.80 mg/L in the sample for SA-NAS. The molar absorptivity was calculated to be 1.70×10⁴ L mol^-1 cm^-1 for SA-1-naphthol and 1.66×10⁴ L mol^-1 cm^-1 for SA-NAS. The detection limits were found to be 0.014 and 0.016 mg/L for SA-1-naphthol and SA-NAS, respectively. The preconcentration factors were 8 and 6 for SA-1-naphthol and SA-NAS, respectively. Replicate determinations of seven sample solutions containing 6.6 μg of nitrite for SA-1-naphthol and 5.3 μg of nitrite for SA-NAS gave mean absorbances of 0.486 and 0.382 with relative standard deviations of 0.49 and 0.58%, respectively. Interferences due to various foreign ions have been studied and the method has been applied to the determination of 27–65 μg/L levels of nitrite in natural waters. The recovery and relative standard deviation for water samples were 98–102% and 0.49–0.58% for both systems. Received: 1 December 1995/Revised: 22 April 1996/Accepted: 22 April 1996     

Miscibility of phosphatidylcholine and sulphatide in monolayers

Compression isotherms of mixed monolayers of phosphatidylcholine (PC) and sulphatides (S) were carried out, with the lipids being spread on subphases of different pH and different ionic composition. Several common trends can be observed. All monolayers when compressed reach collapse. Collapse pressures are directly related to the molar fractions of the lipids in the monolayer. Miscibility is not ideal, in general, and is independent on the surface pressure of the monolayer, the mixed monolayers being always (with some exceptions) in a more expanded state than those of the pure components. The thermodynamic parameters corresponding to the mixing interactions have been calculated at the molar composition that gives a maximum deviation from ideality.     

Spectroscopic studies of the stability of monolayers of 2-docosylamino-5-nitropyridine at the air/water interface

Monolayers of 2-docosylamino-5-nitropyridine (DCANP) at the air/water interface were investigated by UV/Vis spectroscopy. The combination of this method with the classic constant-area relaxation technique yields insight into the longtime stability and the collapse behavior of monolayers. We have demonstrated that monolayers of DCANP are certainly stable under standard deposition conditions. At surface pressures above 20 mN/m monolayer instabilities lead to the formation of a three-dimensional head-to-head multilayered structure.     

Analytical characterization of manganese in rainwater and snow samples

 Seven rain and 2 snow samples collected in October and November 1993 were analyzed by GFAAS. Manganese concentrations ranging from 0.3 to 11.3 μg/L were found. pH-values (4.04–4.89, mean 4.52) and redox potentials (528–665 mV, mean 581 mV) were additionally determined. This is important with respect to a qualified specification of Mn in atmospheric samples. A 0.45 μm-filtration of the samples prior to GFAAS-analysis showed the same manganese concentrations in the filtrate as in the unfiltered samples. Hence, the amount of solid Mn species in the investigated samples is negligible. For an examination of the oxidation state of Mn some investigations concerning the stability of Mn(III) were carried out. They indicated that Mn(III) is unstable under atmospheric conditions. For checking whether all the manganese in rainwater exists as Mn(II), a sensitive IC method for the detection of Mn(II) was developed. By adapting a photometric procedure based on the oxidation of Leucomalachite Green (LMG) to Malachite Green (MG) by permanganate (MnO^- ₄) to the IC-flow-through-system, a limit of determination of 1 μg/L Mn(II) could be achieved. A comparison of the IC measurements with GFAAS-results of the filtered samples showed agreeing results. Thus, manganese in rainwater and snow exists entirely as soluble Mn(II). Received: 24 May 1996/Revised: 7 July 1996/Accepted: 14 July 1996     

Analytical characterization of manganese in rainwater and snow samples

 Seven rain and 2 snow samples collected in October and November 1993 were analyzed by GFAAS. Manganese concentrations ranging from 0.3 to 11.3 μg/L were found. pH-values (4.04–4.89, mean 4.52) and redox potentials (528–665 mV, mean 581 mV) were additionally determined. This is important with respect to a qualified specification of Mn in atmospheric samples. A 0.45 μm-filtration of the samples prior to GFAAS-analysis showed the same manganese concentrations in the filtrate as in the unfiltered samples. Hence, the amount of solid Mn species in the investigated samples is negligible. For an examination of the oxidation state of Mn some investigations concerning the stability of Mn(III) were carried out. They indicated that Mn(III) is unstable under atmospheric conditions. For checking whether all the manganese in rainwater exists as Mn(II), a sensitive IC method for the detection of Mn(II) was developed. By adapting a photometric procedure based on the oxidation of Leucomalachite Green (LMG) to Malachite Green (MG) by permanganate (MnO^- ₄) to the IC-flow-through-system, a limit of determination of 1 μg/L Mn(II) could be achieved. A comparison of the IC measurements with GFAAS-results of the filtered samples showed agreeing results. Thus, manganese in rainwater and snow exists entirely as soluble Mn(II). Received: 24 May 1996/Revised: 7 July 1996/Accepted: 14 July 1996     

Determination of Fe-55 and Ni-63 using semi-preparative ion chromatography – a feasibility study

 The feasibility of semi-preparative high-performance ion chromatography (HPIC) for the analysis of the activation products Fe-55 and Ni-63 in samples of different sources was studied. A mixed anion/cation exchanger column was applied to the separation of Fe(III), Ni(II) and Co(II) using 1 mmol/L PDCA-solution as an eluent. After fractionation of the eluate, Fe(III)- and Ni(II)-fractions were analysed by low-level liquid scintillation counting (LSC). The method was applied to the analysis of corrosion products originating from the primary circuit of a shut-down nuclear power plant. The limits of detection are dependent on the loading of metal ions on the column and are therefore given in terms of specific activities. For a typical sample composition they were found to be 0.011 Bq per mg Fe for Fe-55 and 0.054 Bq per mg Ni for Ni-63. An assessment of semi-preparative HPIC connected to off-line and on-line LSC as compared with standard radiochemical procedures is given. Received: 10 May 1996 / Revised: 2 July 1996 / Accepted: 6 July 1996     

Optimization of the reduction of Se(VI) to Se(IV) in a microwave oven

 Parameters for the reduction of Se(VI) to Se(IV) in HCl medium by heating in a microwave oven have been optimized. The reduction resulted to be quantitative applying 100% power, corresponding to 600 W heating for 2 min in 6 mol/L or for 3 min in 4 mol/L HCl. The behavior of selenomethionine and selenocystine under the optimized reduction conditions was studied in order to evaluate a possible interference of these selenium species in the determination of Se(VI). The final determination of Se(IV), and Se(VI) were done by hydride generation-atomic absorption spectrometry. The analytical merits of the method are reported. The method was applied to the selective determination of Se(IV), and Se(VI) in spiked river and lake water. Received: 6 December 1996/Revised: 1 April 1997/Accepted: 3 April 1997     

Properties of monolayers of crown ethers bearing an azo and azoxy group in the macrocycle

Two methods are described for preparing monomolecular layers of crown ethers with an azo or azoxy group in the macrocycle. When the molecules used to build the monolayer are soluble in aqueous solutions, adsorptive preconcentration on mercury electrodes was used to prepare the monolayer coating. The monolayer was electroactive due to the presence of the azo or azoxy unit in the molecules. Monolayers of crown ethers bearing an azo group in the macrocycle were shown to recognize alkali metal cations present in the solution. Changes of the parameters of the voltammetric reduction peaks - peak potential and peak width, served as an indication of specific interactions of the monolayer of 13-membered and 16-membered azocrown ethers with Na⁺ and K⁺ cations, respectively.The monolayers capable of recognizing cations have also been prepared on the aqueous solution-air interface, using the Langmuir technique. In this approach, amphiphilic derivatives of the azocrowns were synthesized and the monolayer has been assembled on the subphase containing metal cations. Binding of the cation by the macrocycle has a stabilizing effect on the monolayer and higher collapse pressures are achieved than on the pure water subphase. The monolayer was transferred from the air-water interface on the solid substrate using the Langmuir-Blodgett technique. Thin mercury film electrodes on the Ag substrate, or An films evaporated on glass slides were employed as the electrode substrates. The former gave monolayer modified electrodes of higher stability.Presented at the Sixth International Seminar on Inclusion Compounds, Istanbul, Turkey, 27–31 August, 1995.     

Simultaneous voltammetric determination of molybdenum and copper in biological samples

 A selective, sensitive and reliable voltammetric method for the simultaneous determination of Cu and Mo is developed. Both metals form complexes with 8-hydroxyquinoline (oxine). Mo gives two reduction peaks with oxine in acidic chloride media at −0.52 V and −0.58 V, while copper exhibits only one at −0.14 V. Common heavy metals do not interfere at all. The limit of detection is 0.29 ng/ml for Mo and 0.14 ng/ml for Cu after preconcentration on the hanging mercury drop electrode for 30 s at −0.2 V. The R.S.D. at a concentration level of 10 ng/ml is 3.8% for Cu and 5.3% for Mo. The method is applied to different biological samples. Received: 15 January 1996/Revised: 11 April 1996/Accepted: 16 April 1996     

Distillation, on-line RP C18 preconcentration and HPLC-UV-PCO-CVAAS as a new combination for the determination of methylmercury in sediments and fish tissue

 Distillation as a way of sample digestion has been combined with on-line RP C18 preconcentration and HPLC-UV-PCO-CVAAS (high performance liquid chromatography – ultra violet – post column oxidation – cold vapour atomic absorption spectrometry) for the determination of methylmercury at background levels in sediments, soils and fish tissue. To prove the accuracy of this method, it was applied to sediment and fish tissue reference materials. The results correspond with the reference values within their error ranges. Excellent recoveries (92–95%) were obtained for the sediment samples by means of the standard addition method. The standard deviations of the sediment samples were within an acceptable range (7.2–12.5%), those of the fish samples were substantially lower (3.4–5.0%). The detection limit is 0.04 ng/g for 1 g sample weight. Received: 23 November 1995/Revised: 16 April 1996/Accepted: 20 April 1996     

Electrochemical properties of mixed self-assembled monolayers on gold electrodes containing mercaptooctylhydroquinone and alkylthiols

Mixed self-assembled monolayers of 2-(mercaptooctyl)hydroquinone (QH2) and alkylthiols were formed on gold electrodes in EtOH and the redox process of the hydroquinone moiety of QH2 was characterized by cyclic voltammetry (CV) in 0.1 M H(2)SO(4). The monolayers were formed at a series of QH2:alkylthiol ratios and the QH2:alkylthiol ratio in solution was compared to the electrochemical response from QH2 in the obtained monolayer. Mixed monolayers of QH2 with hexylthiol, dodecylthiol, and octadecylthiol were studied. The length of the alkylthiol is crucial for the electrochemical response from QH2 in the monolayer. The total concentration of thiols during monolayer formation and incubation times were also studied and low concentrations of < 2.5 mM and long incubation times gave rise to lower peak separation, lower peak half widths in the CVs of the mixed monolayers, and lower background current. The stability of a pure QH2 monolayer and a 1:4 QH2:hexylthiol monolayer toward high potentials of up to 1.5 V versus Ag/AgCl was also studied and it was observed that the mixed monolayer is significantly more stable than the pure QH2 monolayer.