Interfacial photochemical tautomerization in polyacryloylacetone monolayers

 The surface organization of the enol units of polyacryloylacetone (PAA) monolayer at the air–water interface is examined using surface pressure and surface potential measurements and theoretical calculations based on molecular models. The mechanism and kinetics of the photochemical enol–keto tautomerization is studied by measuring the increase of the surface area at constant surface pressure. The effect is due to the increase of the area per monomer units during the consecutive enol-to-keto photoconversion and slow interfacial reorganization of these forms to a more favorable state. Received: 9 March 1998 Accepted: 29 May 1998     

Chain dependence in phospholipid interactions: A thermodynamic study of mixed monolayers

 The interactions between three of the major phospholipids in biomembranes, distearoylphos-phatidylethanolamine, distearoylphosphatidylcholine and sphyngomyelin, forming monolayers at the air–water interface, are studied. Following the Goodrich [1] thermo-dynamic formulation, a quantitative analysis on these interactions is carried out. The general conclusion reached is that, depending on the molecular structure of the lipid and the experimental temperature, significant interactions occur between lipid molecules. One hypothesis is the possibility that, the usual miscibility analysis, in mixed monolayers, could be inadequate to provide information on the interactions in the systems which the hydration forces are significant, is advanced. Received: 30 May 1997 Accepted: 5 October 1997     

Enzymatic hydrolysis of poly(D, L-lactide) spread monolayers by cutinase

 The enzymatic hydrolysis of a model poly(D,L-lactide) by cutinase was studied by using a barostat surface balance. A theoretical approach based on the adaptation of the Michaelis–Menten scheme at the interface and a process of random fragmentation of the macromolecules was developed. The role of the interfacial organisation of the reaction products was discussed. Hydrolysis rate constant values and the specific activity were estimated and compared with those obtained for the hydrolysis of lipid monolayers. The process of fragmentation of the interfacial polymer structures was visualised by using AFM imaging. Received: 19 August 1996 Accepted: 13 December 1996     

Evaluation of molecular structure in Langmuir monolayers of zinc stearate and zinc 12-hydroxystearate by IR external reflection spectroscopy

 In situ polarized Fourier transform IR external reflection spectra of Langmuir monolayers of zinc stearate and zinc 12-hydroxystearate on a water surface were recorded for various surface areas, and their molecular structures were estimated. In the zinc stearate monolayer, the wavenumbers and the absorbances of the antisymmetric and symmetric methylene stretching bands did not change during monolayer compression, which means that orientational and conformational changes of the hydrocarbon chain did not occur. However, wavenumber changes of the antisymmetric and symmetric carboxylate stretching bands were observed during surface compression. The change in the binding nature of the zinc cation to the carboxylate group was speculated. Moreover, it was elucidated that the structure of the hydrocarbon chain in the zinc 12-hydroxystearate monolayer was different from that in the zinc stearate monolayer. Received: 21 March 2001 Accepted: 6 July 2001     

Quantum mechanics and mixed quantum mechanics/molecular mechanics simulations of model nerve agents with acetylcholinesterase

 The accurate modeling of biological processes presents major computational difficulties owing to the inherent complexity of the macromolecular systems of interest. Simulations of biochemical reactivity tend to require highly computationally intensive quantum mechanical methods, but localized chemical effects tend to depend significantly on properties of the extended biological environment – a regime far more readily examined with lower-level classical empirical models. Mixed quantum/classical techniques are gaining in popularity as a means of bridging these competing requirements. Here we present results comparing two quantum mechanics/molecular mechanics implementations (the SIMOMM technique of Gordon et al. as implemented in GAMESS, and the ONIOM technique of Morokuma et al. found in Gaussian 98) as performed on the enzyme acetylcholinesterase and model nerve agents. This work represents part of the initial phase of a DoD HPCMP Challenge project in which we are attempting to reliably characterize the biochemical processes responsible for nerve agent activity and inhibition, thereby allowing predictions on compounds unrelated to those already studied. Received: 10 October 2001 / Accepted: 13 November 2002 / Published online: 1 April 2003 Contribution to the Proceedings of the Symposium on Combined QM/MM Methods at the 222nd National Meeting of the American Chemical Society, 2001 Correspondence to: M. M. Hurley e-mail: hurley@arl.army.mil     

Adsorption of block polyampholyte micelles in monolayers at the silicon water interface

 The adsorption of the diblock polyampholyte poly (methacrylic acid)-block-poly((dimethylamino)ethyl methacrylate) from aqueous solution on silicon substrates was investigated as a function of polymer concentration and pH. Dynamic light scattering and electrokinetic measurements were used to characterize the polyampholyte in solution. The amount of polymer adsorbed was determined by ellipsometry and lateral structures of the polymer layer were investigated by scanning force microscopy. The amount of polymer adsorbed was found to be strongly influenced by the pH of the polymer solution, while the size of the polyampholyte micelles adsorbed on the surface was hardly affected by pH during adsorption. From investigations by scanning force microscopy well-seperated micelles were seen in the dried monolayers adsorbed directly from solution. The structures at the surface are correlated to structures in solution, and the adsorbed amount depends on the relative charge of the micelles and the surface. Received: 13 September 1999 Accepted in revised form: 8 December 1999     

Study of the interaction and activation of lipase from Pseudomonas fluorescens in surfactant monolayers and precipitates

The activation of lipase from Pseudomonas fluorescens (PFL) upon its immobilization in surfactant coprecipitates (hexadecane-1,2-diol (HDD), cetyl alcohol (CetOH), N-cetylacetamide (CetAA), and cetylamine (CetNH₂)) organized in monolayers at the interface were studied by the Langmuir—Blodgett monolayer technique. Incorporation of the enzyme into surfactant monolayers at the surface pressure = 10 mN m^–1 results in an apparent increase in the area per molecule. In the series of noncharged surfactants CetOH—HDD—CetAA, this effect increases in proportion to the amount of the enzyme incorporated in the monolayer. The catalytic activity of the lipase—surfactant coprecipitates in an organic solvent as regards esterification increases in the same sequence, indicating similarity of the interaction of lipase with surfactant monolayers and coprecipitates. For = 10 mN m^–1, the CetNH₂ monolayer with liquid-expanded state incorporates the largest amount of the enzyme (PFL : CetNH₂ = 1 : 290); the CetOH monolayer, which exists in the condensed state under the same conditions, incorporates the smallest amount (PFL : CetOH = 1 : 1700). The hydrolytic activity of PFL in mixed monolayers with surfactants increases 1.5—11-fold; the esterification activity in surfactant coprecipitates, 1.6—9-fold. The lipase activation effects are explained by facilitated transport of substrates into mixed monolayers and surfactant—enzyme precipitates in aqueous and organic media, respectively.     

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     

Electrophoretic behaviour and viscosities of metal oxides in mixed surfactant systems

 The electrokinetic behavior and viscosity of anatase and alumina in mixed-surfactant solutions were investigated. Sodium dodecylsulfate and nonionic polyoxyethylene ethers were investigated as model surfactants. Pure nonionic surfactants adsorbed on anatase and coated the particles, so that the zeta potential was nearly zero near the critical micelle concentration of surfactant. At higher surfactant concentrations, an increase in the zeta potentials was observed, suggesting a change in the microstructure of the adsorbed layer. Addition of nonionic surfactant to positively charged anatase and alumina with some preadsorbed sodium dodecylsulfate reversed the surface charge of the oxide to negative, indicating enhanced coadsorption of the anionic surfactant. At higher concentrations of the nonionic surfactant, the charge reversed back to positive. Nonionic surfactants did not reverse the surface charge of these oxides in the absence of the anionic surfactant. Coenhanced adsorption of nonionic and anionic surfactants was used to stabilize alumina at the isoelectric point, where neither surfactant adsorbed appreciably on its own. These results suggest a dramatic change in conformation of the surfactant chains in mixed systems. Further explanation and justification of the proposed changes in adsorbed surfactant conformation require spectroscopic evidence. Received: 12 March 1997 Accepted: 22 July 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     

Recent advances in quantum mechanical/molecular mechanical calculations of enzyme catalysis: hydrogen tunnelling in liver alcohol dehydrogenase and inhibition of elastase by α-ketoheterocycles

 Hybrid quantum mechanical (QM)/molecular mechanical (MM) calculations are used to study two aspects of enzyme catalysis, Kinetic isotope effects associated with the hydride ion transfer step in the reduction of benzyl alcohol by liver alcohol dehydrogenase are studied by employing variational transition-state theory and optimised multidimensional tunnelling. With the smaller QM region, described at the Hartree–Fock ab initio level, together with a parameterised zinc atom charge, good agreement with experiment is obtained. A comparison is made with the proton transfer in methylamine dehydrogenase. The origin of the large range in pharmacological activity shown by a series of α-ketoheterocycle inhibitors of the serine protease, elastase, is investigated by both force field and QM/MM calculations. Both models point to two different inhibition mechanisms being operative. Initial QM/MM calculations suggest that these are binding, and reaction to form a tetrahedral intermediate, the latter process occurring for only the more potent set of inhibitors. Recieved 3 October 2001 / Accepted: 6 September 2002 / Published online: 31 January 2003 Contribution to the Proceedings of the Symposium on Combined QM/MM Methods at the 222nd National Meeting of the American Chemical Society, 2001 Correspondence to: I. H. Hillier Acknowledgements. We thank EPSRC and BBSRC for support of the research and D.G. Truhlar for the use of the POLYRATE code.     

Specific adsorption of anions in the course of underpotential and bulk depositions and alloy formation of cadmium on a smooth gold support in acid medium

 The specific adsorption of ³⁶Cl-labelled Cl⁻ ions and ³⁵S-labelled HSO₄ ⁻ ions was studied in 1 mol dm⁻³ HClO₄ supporting electrolyte in the presence of Cd²⁺ ions at a gold support over a wide potential range corresponding to electrodeposition, alloy formation, underpotential deposition of Cd species and existence of an adatom-free surface. The distinct sections in the potential dependence of the adsorption of anions together with the potential versus time curves obtained under open circuit conditions reflect the changes in the state of the electrode surface, the dissolution of the bulk Cd phase and the slow elimination of Cd species from the Cd/Au alloy. Received: 16 March 1999 / Accepted: 5 May 1999     

Aggregation effects on evaporation and the air/water interface of dodecyltrimethylammonium hydroxide solutions

 The system dodecyltrimethylammonium hydroxide (DTAOH)–water was studied by surface tension, ion-selective electrodes and evaporation in an electrobalance. Results confirmed earlier conclusions about a stepwise aggregation mechanism in DTAOH solutions. The aggregation process started at a total concentration C _T=(2.51±0.10)×10^-4 mol dm^-3) which probably corresponds to the formation of dimers. At C _T= (1.300±0.041)×10^-3 mol dm^-3 there was a change in the surface and evaporation behavior, corresponding to the formation of small, fully ionized aggregates which grew with increasing concentration. At C _T= (1.108±0.010)×10^-2 mol dm^-3 the formation of true micelles with hydroxide counterions in the Stern layer did not change significantly the evaporation and adsorption behavior. This means that between this concentration and C _T=(3.02±0.28)× M28.8n10^-2 mol dm^-3, the changes in structure were gradual. At the latter concentration there was a sudden change in the monolayer state at the air/water interface, with a strong surfactant desorption, and a major change in evaporation behavior. The changes are compatible with the formation of few, large aggregates reducing the total concentration of kinetically independent solute units, which in turn increased the activity of the solvent. This phenomenon is in agreement with literature information. The reduction in the evaporation rate of water was mainly due to the reduction of the water activity, caused by colligative effects. The reduction of the effective area available for evaporation had only a slight effect in water evaporation. Received: 9 January 1997 Accepted: 19 October 1997     

Structural and catalytic aspects of cutinase in w/o microemulsions

 Structural and catalytic properties of cutinase were studied in bis(2-ethylhexyl) sodium sulfo-succinate (AOT)-isooctane microemulsion systems. The effect of the water content of the microemulsions on the cutinase activity on an esterification reaction of lauric acid with pentanol showed that cutinase followed a bell-shaped profile presenting a maximum at w _o=9, with w _o=[H₂O]/[AOT]. Kinetic studies allowed the determi-nation of the apparent parameters K _m and V _max. Electron paramagnetic resonance (EPR) spectroscopy studies of active site labeled cutinase in microemulsions with varying w _o values showed that in all microemulsions, the mobility of the label is higher than in the aqueous solution. Furthermore, it was found that the maximum of the enzyme activity did not correspond to a reduced active site mobility. Up to w _o=9 there was an increase of both activity and active site mobility. As the water content of the system became higher, the mobility of the bound spin label further increased whereas the enzymatic activity dropped considerably. Received: 20 December 1996 Accepted: 24 February 1997     

The effect of counterions on the surface adsorption and micelle formation in mixed surfactant systems of dodecyl sulfates

 The surface tension of the aqueous solutions of sodium dodecyl sulfate (SDS) and tetramethylammonium dodecyl sulfate (TMADS) was measured as a function of total molality of the surfactants at fixed composition of TMADS at 298.15 K under atmospheric pressure. The phase diagrams of adsorption and of micelle formation, the activity coefficients, and the excess Gibbs energy were calculated to estimate the deviation from the ideal mixing quantitatively. The preferential adsorption and the micelle formation for TMA⁺ to Na⁺ is attributable to some extent to the hydrophobicity of the methyl groups of TMA⁺. The composition of TMA⁺ in the micelle is larger than that in the adsorbed film at equilibrium. That is, a larger hydrated counterion is more likely to exist in the micelle than in the adsorbed film owing to geometrical benefit. The negative values of the excess Gibbs energy of the adsorbed film and of the micelle arise from the positive ones of the excess entropy greater than that of excess enthalpy. The counterions of very similar size are mixed ideally in the micelle and the size effect appears sensitively in the adsorbed film. Received: 23 May 2001 Accepted: 16 July 2001     

Stereoselectivity and competing reactions as studied by lipase-catalyzed esterifications in aqueous lecithin-based gelatin gels

 The enzyme catalyzed conversion of R/S-(±)-2-octanol with hexanoic acid to R/S-(±)-2-octyl hexanoate has been studied in different microenvironments and in the presence of the competing substrate ethanol. The reactions were performed in various gels made from aqueous gelatin solutions and liposome dispersions or isotropic liquid solutions, with or without oil and ethanol. The lipase Candida sp. (SP 525) was dissolved in the dispersions or solutions stabilized by the naturally occurring zwitterionic surfactant soybean lecithin. The sectioned porous gel was immersed in hexane containing 0.33 mol dm^-3 of racemic 2-octanol and hexanoic acid. Since ethanol acts both as a substrate and as a part of the gel it is of fundamental interest to know the phase behaviour of the used systems. Partial phase diagrams for the systems ethanol–water–soybean lecithin and ethanol/water (7:3)–oil–soybean lecithin were determined at 298.2 K. The oil was either castor oil or hexadecane. The conversion of R-2-octyl hexanoate was about 0.45 when no or small amounts of ethanol was present, but decreased considerably with high amounts of ethanol present and ethyl hexanoate became the main product. Hydrolysis of R-2-octyl hexanoate was favoured in the latter systems and hexanoic acid formed was immediately esterified to ethyl hexanoate. The conversion of R-2-octyl hexanoate and ethyl hexanoate depends only on the ethanol content present in the systems and is thus independent of the environment of the enzyme. However, the chiral esters synthesized from racemic 2-octanol and hexanoic acid showed high optical purities regardless of the ethanol content. Received: 1 July 1996 Accepted: 30 August 1996     

Flow-Injection Spectrophotometric Determination of Diclofenac or Mefenamic Acid in Pharmaceuticals

 Two flow injection (FI) spectrophotometric methods are proposed for the determination of diclofenac (DCF) or mefenamic acid (MF) in bulk samples and pharmaceuticals. Both methods are based on the reaction of DCF or MF with potassium ferricyanide in a sodium hydroxide medium. The absorbance of the orange products obtained is measured at 455 nm for DCF and 465 nm for MF. The corresponding calibration graphs are linear over the range 0.20–20.0 mg L⁻¹ for DCF and 1.00–100 mg L⁻¹ for MF, while the limits of detection were 0.05 and 0.18 mg L⁻¹, respectively. Received March 27, 2000. Revision November 15, 2000.     

Preparation of Hydroxy-Substituted Hexahydrophthalazinones from Cyclohexane- and Norbornanelactones or Ketallactones

Summary.  Cyclohexane- and norbornanelactones (1, 5) as well as ketallactones (3, 7) and their hydrolytic products (2, 4a,b, 8) react with hydrazine hydrate to yield the hydroxy-substituted hexahydrophthalazinones 9–12. In the cyclizations, the configurations of the bridgehead atoms remain unaltered, and the hydroxy group retains its original position in the hydrazinolysis of 5 and 7. The compounds were characterized by IR, ¹H, and ¹³C NMR spectroscopy. Received September 3, 2001. Accepted October 8, 2001     

Enrichment in γ-linolenic acid of acylglycerols by the selective hydrolysis of borage oil

Selective hydrolysis of borage oil byCandida rugosa lipase immobilized on microporous polypropylene was carried out in an iso-octane-water two-phase system in order to obtain glycerides rich in γ-linolenic acid (GLA). Lipase was immobilized on hydrophobic microporous polypropylene supports by physical adsorption, γ-linolenic acid content in the unhydrolyzed acylglycerols could be raised to 51.7 mol% from an initial content of 23.6 mol% in borage oil with a yield of 59%. A simplified kinetic model was proposed for this selective hydrolysis. The Michaelis constantK _M and the maximal-rate constantV _max are 0.107M and 393.9 U/mg-protein, respectively. Product inhibition with a dissociation constant of the enzyme-product complexK _I = 25 mM was confirmed. Some properties of the immobilized lipase were also examined.     

Investigation on the basic hydrolysis of crystal violet in mixed reverse micelles formed with AOT and nonionic surfactants

 The kinetics and thermodynamics of the basic hydrolysis of crystal violet (CV) in mixed reverse micelles formed with anionic surfactant AOT and nonionic surfactants have been investigated. It was found that the mixed reverse micelles had inhibitory effects on CV hydrolysis compared with the normal aqueous solution, and the equilibrium constant K of the reaction in mixed reverse micellar systems is smaller than that in pure water. The influence of water content and surfactant composition in reverse micelles on the second-order rate constant k ₁ of the positive reaction, on the first-order rate constant k _-1 of the reverse reaction, as well as on the equilibrium constant K of the reaction has been studied, and the results obtained were interpreted in terms of the nature of surfactants and the properties of microenvironment where the reaction took place. Received: 24 October 1997 Accepted: 18 March 1998