Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

Determination of molecular weight and molecular sizes of polymers by high temperature gel permeation chromatography with a static and dynamic laser light scattering detector

Flow-mode static and dynamic laser light scattering (SLS/DLS) studies of polymers, including polystyrene, polyethylene, polypropylene and poly(dimethylsiloxane) (PDMS), in 1,2,4-trichlorobenzene (TCB) at 150 degreesC were performed on a high temperature gel permeation chromatography (GPC) coupled with a SLS/DLS detector. Both absolute molecular weight (M) and molecular sizes (radius of gyration, R-g and hydrodynamic radius, R-h) of polymers eluting from the GPC columns were obtained simultaneously. The conformation of different polymers in TCB at 150 degreesC were discussed according to the scaling relationships between R-g, R-h and M and the rho-ratio (p = R-g/R-h). Flow-mode DLS results of PDMS were verified by batch-mode DLS study of the same sample. The presented technique was proved to be a convenient and quick method to study the shape and conformation of polymers in solution at high temperature. However, the flow-mode DLS was only applicable for high molecular weight polymers with a higher refractive index increment such as PDMS.

Species of lanthanum in Wistar rat liver

The metabolic accumulation and species of lanthanum in Wistar rat liver were investigated by ICP-MS, gel exclusion chromatography and ultrafiltration after the rats were fed by low dose of lanthanum for a long time. It was found that the content of La in the liver increased regularly with arise of dose and time of drug delivery. After the administration was stopped for a certain time a part of lanthanum in the liver Tvas metabolized, but;the metabolic rate was very slow, The lanthanum in rat liver was distributed in the soluble protein with molecular weight: of more than 60000 mostly. Rare Earth existed in the six elution peaks separated by Sephacryl S-200. The amount of lanthanum in the first elution fraction is the largest, which was 88 percent in the whole content of lanthanum in proteins with molecular weight more than 60000.

A new method of measuring alcohol clusters in polyimide membrane: combination of inverse gas chromatography with equilibrium swelling

A new method of measuring the mean size of solvent clusters in swollen polymer membrane is presented in this paper. This method is based on a combination of inverse gas chromatography (IGC) and equilibrium swelling. The mechanism is that weight fraction activity coefficient of solvent in swollen polymer is influenced by its clusters size. The mean clusters size of solvent in swollen polymer can be calculated as the quotient of the weight fraction activity coefficient of clustering system dividing the weigh fraction activity coefficient of non-clustering system. In this experiment, the weigh fraction activity coefficient of non-clustering system was measured with IGC. Methanol, ethanol and polyimide systems were tested with the new method at three temperatures, 20, 40, and 60degreesC. The mean clusters size of methanol in polyimide was five, four, and three at each temperature condition, respectively. Ethanol did not form clusters (the mean clusters size was one). In contrast to the inherent narrow temperature range in DSC, XRD, and FTIR methods, the temperature range in IGC and equilibrium swelling is broad. Compared with DSC. XRD. and FTIR, this new method can detect the clusters of solvent-polymer system at higher temperature.

Electrospray ionization mass spectrometry of cyclodextrin complexes with amino acids in incubated solutions and in eluates of gel permeation chromatography

1:1 complexes of beta-cyclodextrin (CD) with three amino acids (Gly, Phe and Trp) have been detected as ions in the gas phase using infusion positive and negative ion electrospray ionization mass spectrometry (ESI-MS). In contrast with the positive ion ESI mass spectra of simple aqueous solutions, the aggregates and adducts usually formed in the ESI process did not appear in the positive ion ESI spectra of solutions buffered with ammonium acetate (NH4Ac), even at higher analyte concentrations, These studies suggest that addition of buffer and/or use of a low analyte concentration should be used to overcome formation of aggregates and metal ion adducts in such mass spectrometry studies. Also, the deprotonated complexes are dissociated by collision induced dissociation (CID) to form an abundant product ion, the deprotonated CD, requiring transfer of a proton to the amino acid carboxyl group, To understand formation of complexes in the gas phase, gel permeation chromatography (GPC) was used to separate free amino acids (AAs) from complexes in an incubated solution. The ESI mass spectra of the GPC fractions show the presence of 1:1 complexes of both CD-aromatic amino acids and CD-aliphatic amino acids. Compared with CD-aliphatic amino acid complexes, CD-aromatic amino acid complexes appear to be destabilized in the gas phase, possibly because the hydrophobic interaction which binds the aromatic group of amino acids in the CD cavity in solution may become repulsive when solvent evaporates from the droplets during the electrospray process, whereas those complex ions formed as proton bound dimers are stabilized by electrostatic forces, the major binding force for such complexes in the gas phase. In addition, the GPC technique coupled with off-line ESI-MS can rapidly separate CD complexes by size, and provides some information on the character of the complexes in solution. (C) 1998 John Wiley & Sons, Ltd.

Separation and determination of organogermanium compounds by ion chromatography

For nearly three decades, organogermanium compounds have become increasingly of interest owing to their extensive physiological and pharmaceutical activity. In this paper, two new high performance ion chromatographic methods for separation and determination of three kinds of organogermanium compounds beta-carboxyethylgermanium sesquioxide (I), beta-(alpha-methyl)-carboxyethylgermanium sesquioxide (II) and d-(beta-carboxyethyl)germanium hydroxide (III) were proposed. A Dionex DX-300 ion chromatograph equipped with a Dionex FED-II pulsed electrochemical detector (conductivity mode), and a Dionex AI-450 chromatography workstation was employed. The separation was achieved by using ion-exchange or ion-exclusion mechanism. The detection limits(S/N=3, expressed as germanium) for the three compounds were all below sub- mu g/mL level. The methods have been applied to the analysis of tonic oral drinks, and the average recoveries for the three compounds range from 95 - 108%. The results obtained were in agreement with those of hydride generation atomic fluorescence spectrometry (HG-AFS).

Linear solvation energy relationships regarding sorption and retention properties of hydrophobic organic compounds in soil leaching column chromatography

The capacity factors of a series of hydrophobic organic compounds (HOCs) were measured in soil leaching column chromatography (SLCC) on a soil column, and in reversed-phase liquid chromatography on a C-18 column with different volumetric fractions (phi) of methanol in methanol-water mixtures. A general equation of linear solvation energy relationships, log(XYZ) = XYZ(0) + mV(1)/100 + spi* + bbeta(m) + aalpha(m), was applied to analyze capacity factors (k'), soil organic partition coefficients (K-oc) and octanol-water partition coefficients (P). The analyses exhibited high accuracy. The chief solute factors that control log K-oc, log P, and log k' (on soil and on C-18) are the solute size (V-1/100) and hydrogen-bond basicity (beta(m)). Less important solute factors are the dipolarity/polarizability (pi*) and hydrogen-bond acidity (alpha(m)). Log k' on soil and log K-oc have similar signs in four fitting coefficients (m, s, b and a) and similar ratios (m:s:b:a), while log k' on C-18 and log P have similar signs in coefficients (m, s, b and a) and similar ratios (m:s:b:a). Consequently, log k' values on C-18 have good correlations with log P (r > 0.97), while log k' values on soil have good correlations with log K-oc (r > 0.98). Two K-oc estimation methods were developed, one through solute solvatochromic parameters, and the other through correlations with k' on soil. For HOCs, a linear relationship between logarithmic capacity factor and methanol composition in methanol-water mixtures could also be derived in SLCC. (C) 2002 Elsevier Science Ltd. All rights reserved.

Size-dependent inelastic behavior of particle-reinforced metal-matrix composites

The strengthening behavior of particle-reinforced metal-matrix composites (MMCp) is primarily attributed to the dislocation strengthening effect and the load-transfer effect. To account for these two effects in a unified way, a new hybrid approach is developed in this paper by incorporating the geometrically necessary dislocation strengthening effect into the incremental micromechanical scheme. By making use of this hybrid approach, the particle-size-dependent inelastic deformation behavior of MMCp is given. Some comparisons with the available experimental results demonstrate that the present approach is satisfactory.

Kinetic and Size Control of Polystyrene and Polyacrylic Octadecyl Ester Lattices Via Polymerization in O/W Microemulsions

The kinetic studies of the acrylic octadecyl ester and styrene polymerization in microemulsion systems, (1) cetyl pyridine bromide (CPDB)/t-butanol/styrene/water; (2) CPDB/t-butanol/toluene + acrylic octadecyl ester (1:1, w/v)/ water; (3) cetyl pyridine bromide/styrene/formamide, were made by using dynamic laser light scattering techniques (DLS). The mechanisms of nucleation of latex particles were discussed. The most possible nucleation location of the styrene and acrylic octadecyl ester microlatex particles in aqueous microemulsion system is in aqueous phase via homogeneous nucleation. Meanwhile, parts of microlatex particles are possibly produced via swollen micelles (microemulsions) and monomer droplets nucleation. On the other hand, the most possible nucleation location of the styrene microlatex particles in nonaqueous microemulsion system is inside monomer droplets. The relationship between the amount of monomer and the size of microlatex was also investigated. It has been found that the size of microlatex particles could be controlled by changing the amount of monomer. (C) 2002 Elsevier Science B.V. All rights reserved.

Effect of Rock Mass Structure and Block Size on the Slope Stability-Physical Modeling and Discrete Element Simulation

This paper studies the stability of jointed rock slopes by using our improved three-dimensional discrete element methods (DEM) and physical modeling. Results show that the DEM can simulate all failure modes of rock slopes with different joint configurations. The stress in each rock block is not homogeneous and blocks rotate in failure development. Failure modes depend on the configuration of joints. Toppling failure is observed for the slope with straight joints and sliding failure is observed for the slope with staged joints. The DEM results are also compared with those of limit equilibrium method (LEM). Without considering the joints in rock masses, the LEM predicts much higher factor of safety than physical modeling and DEM. The failure mode and factor of safety predicted by the DEM are in good agreement with laboratory tests for any jointed rock slope.

Size Effect and Geometrical Effect of Polycrystals and Thin Film/Substrate System in Micro-indentation Test

Micro-indentation test at scales on the order of sub-micron has shown that the measured hardness increases strongly with decreasing indent depth or indent size, which is frequently referred to as the size effect. Simultaneously, at micron or sub-micron scale, the material microstructure size also has an important influence on the measured hardness. This kind of effect, such as the crystal grain size effect, thin film thickness effect, etc., is called the geometrical effect by here. In the present research, in order to investigate the size effect and the geometrical effect, the micro-indentation experiments are carried out respectively for single crystal copper and aluminum, for polycrystal aluminum, as well as for a thin film/substrate system, Ti/Si3N4. The size effect and geometrical effect are displayed experimentally. Moreover, using strain gradient plasticity theory, the size effect and the geometrical effect are simulated. Through comparing experimental results with simulation results, length-scale parameter appearing in the strain gradient theory for different cases is predicted. Furthermore, the size effect and the geometrical effect are interpreted using the geometrically necessary dislocation concept and the discrete dislocation theory. Member Price: $0; Non-Member Price: $25.00

Effect of Particle Size on the Formation of Adiabatic Shear Band In Particle Reinforced Metal Matrix Composites

In this paper, the effect of particle size on the formation of adiabatic shear band in 2024 All matrix composites reinforced with 15% volume fraction of 3.5, 10 and 20 mum SiC particles was investigated by making use of split Hopkinson pressure bar (SHPB). The results have demonstrated that the onset of adiabatic shear banding in the composites strongly depends on the particle size and adiabatic shear banding is more readily observed in the composite reinforced with small particles than that in the composite with large particles. This size dependency phenomenon can be characterized by the strain gradient effect. Instability analysis reveals that high strain gradient is a strong driving force for the formation of adiabatic shear banding in particle reinforced metal matrix composites (MMCp).