以透明质酸为骨架的新型谷胱甘肽过氧化物酶(GPX)模拟酶的制备及性质研究

用修饰法合成以透明质酸为骨架的两种新型GPX模拟酶: 硒化透明质酸SeHA及碲化透明质酸TeHA. 用红外光谱和核磁共振波谱对模拟酶的结构进行研究, 证明其修饰位点位于透明质酸的N-乙酰氨基葡萄糖的—CH2OH. 用二硫代双硝基苯甲酸(DTNB)法测定模拟酶的硒含量为1.2%. 通过模拟酶对3种不同底物过氧化氢(H2O2)、过氧化氢正丁烷(t-BuOOH)和过氧化氢异丙苯(CuOOH)的催化活性的研究结果表明CuOOH为该反应的最佳底物. 研究模拟酶催化谷胱甘肽(GSH)还原3种过氧化物的动力学发现, 反应速率与底物浓度的双倒数曲线均为平行的直线, 说明模拟酶反应的动力学机制与天然GPX相同, 为乒乓机制. 用2,4-二叔丁基甲基苯酚(BHT)法证明了该催化反应为非自由基机理, 且模拟酶不易被碘乙酸抑制.     

A semisynthetic glutathione peroxidase with high catalytic efficiency. Selenoglutathione transferase

Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.     

新型兼具GPX,SOD, CAT活性的水杨醛Schiff碱衍生物的合成

以5-磺基水杨醛为母体, 经氯甲基化和硒化反应引入催化基团-SeH, 此化合物经空气氧化、Schiff碱反应以及锰螯合反应, 最终得到一种新型的水杨醛Schiff碱类谷胱甘肽过氧化物酶(GPX)模拟物. 测定了该模拟物的红外光谱、核磁共振谱、质谱和抗氧化能力. 此模拟物同时兼具超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性, 水溶性好, 保护线粒体免遭氧化损伤能力强, 具有作为药物前体的潜力.     

GSH对两种谷胱甘肽过氧化物酶模拟物活性影响的研究

设计并合成了谷胱甘肽过氧化物酶(GPX)模拟物6A,6A’-二苯胺-6B,6B’-二硒桥联-β-环糊精(6-AnSeCD). 采用双酶偶联法测定GPX的活力结果显示, 6A,6A’-二环己胺-6B,6B’-二硒桥联-β-环糊精(6-CySeCD)催化谷胱甘肽还原H2O2和枯烯H2O2的活力均比6-AnSeCD的高. 为了进一步考察6-CySeCD和6-AnSeCD与GSH之间的相互作用, 进行了分子动力学(MD)模拟和分子对接研究. 结果表明, 与GSH的结合使GPX模拟物的构象发生变化, 这种改变可能是影响桥连GPX模拟物催化活性的关键因素.     

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

IntroductionReactive oxygen species(ROS) are known to de-stroy biomacromolecules and cause cell injury[1]. Un-der normal circumstances, there is a balance betweenthe production of ROS and their destruction. Many dis-eases, such as brain ischemia, tumor, v…     

Apoptotic response to photodynamic therapy versus the Bcl-2 antagonist HA14-1

In this study, murine leukemia L1210 cells were used to compare the effects of photodynamic therapy (PDT) with those of the apoptotic nonpeptidic Bcl-2 ligand ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1). The photosensitizing agent capronyloxy-tetrakis methyloxyethyl porphycene (CPO) was selected from a group of sensitizers previously shown to target the antiapoptotic protein Bcl-2 for photodamage. Like PDT with CPO, HA14-1 caused the rapid activation of procaspase-3, followed by the appearance of an apoptotic morphology within 60 min. Caspase activation after a sublethal dose of either PDT or HA14-1 was enhanced by staurosporine or the bile acid ursodeoxycholic acid. Moreover, PDT promoted procaspase activation and lethality of HA14-1 and vice versa. Effects of PDT versus HA14-1 could not be distinguished on the basis of the reactive oxygen species formation. Both caused the rapid oxidation of 2',7'-dichlorofluorescein. These results are consistent with the hypothesis that Bcl-2 photodamage is a target for some photosensitizing agents.     

Interactions between rotenone and humic acids by means of FT-IR and fluorescence spectroscopies

The aim of this work was to ascertain, on a comparative basis, the compositional, structural and functional differences occurring between three humic acids (HAs), HA S1 (isolated from a Mediterranean brown soil), HA S2 (isolated from a Bavarian brown soil), and HA SR (a Suwannee River standard aquatic HA, purchased from IHSS), and to investigate the influence of their intrinsic properties on the types of binding mechanisms toward the pesticide rotenone. Original HAs and their corresponding HA–rotenone products, obtained by two different interaction protocols, were analyzed for elemental and functional group composition, and spectroscopic techniques, such as Fourier-transform infrared (FT IR) with Fourier self-deconvolution (FSD) and fluorescence both in the single-scan and in three-dimensional modes. The HA S1 sample appeared to be characterized by a greater aromaticity degree and lower polarity with respect to the HA S2, featured by a mixed aromatic/aliphatic character, whereas mainly aliphatic and acidic resulted the HA SR. The data obtained suggested that the low water-soluble, non-polar pesticide rotenone resulted preferentially adsorbed onto HAs by hydrophobic interaction, that was the prevailing mechanism in the order HA S1 > HA S2 >>> HA SR, whereas hydrogen bonds resulted predominant in the opposite order.     

Effect of phenyl substituent on the dimerization of copper(II)-carboxylate in solvent extraction of copper(II) with phenylacetic acid

The effects of the phenyl substituent on the dimerization of copper(II) carboxylate in the solvent extraction of copper(II) with phenylacetic acid using benzene and 1-octanol as a solvent were investigated, at 25 degrees and at the aqueous ionic strength of 0.1M (NaClO(4)). The dimerization of copper(II) phenylacetate has been found to be written as: 2CuA(2)Cu(2)A(4) in 1-octanol, and 2CuA(2)(HA)(2)Cu(2)A(4)(HA)(2) + (HA)(2) in benzene, with the dimerization constants, log K = 2.24 and log K = 4.19, respectively. It was proved that the phenyl group inhibits the formation of the dimeric copper(II) phenylacetate, and its effect is partially shielded by a methylene substituent.     

Periodic ab initio study of structural and vibrational features of hexagonal hydroxyapatite Ca10(PO4)6(OH)2

Structural and vibrational features of hexagonal hydroxyapatite HA [Ca(10)(PO(4))(6)(OH)(2), space group P6(3)] are computed ab initio within a periodic approach using the CRYSTAL03 program and the B3LYP hybrid functional with a Gaussian-type basis set of polarized double zeta quality. Experimental lattice parameters and internal coordinates have been fully optimized and the final structure characterized by means of its band structure, density of states and Mulliken analysis. The full B3LYP harmonic vibrational spectrum of HA at Gamma point has also been computed and compares well with the available experimental IR and Raman data. Nevertheless, the presence of one negative frequency in the computed spectrum shows that, within the hexagonal symmetry imposed by the P6(3) group, the structure is a saddle point. This is at variance with the monoclinic structure (under P2(1)/b space group), which has been computed, with the same approach, to be a minimum of 17 kJ mol(-1) (per unit cell) more stable than the corresponding hexagonal HA structure.     

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.     

Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.     

1-Bis(carboxymethyl)amino-butandion-2,3-dioxim als ambifunktioneller Ligand in Komplexen der 3d-Elemente

1-Bis(carboxymethyl)amino-butanedione-2,3-dioxime as an Ambifunctional Ligand in Complexes of 3d Elements In acid solution 1-bis(carboxymethyl)amino-butanedione-2,3-dioxime H₄A affords octahedral 1,1 complexes M^II(H₂A)(H₂O)_x (M^II = Cu, Ni, Co) coordinating as a tetradentate ligand by the imino diacetic acid group and the oxime group in 2-position. At 4.5 < pH < 8 Ni(H₂A)(H₂O)_x and Cu(H₂A)(H₂O)_x are deprotonated, in addition to Ni(HA)^? the binuclear chelate [Ni₂(HA)₂]^2? is formed. In solution containing a surplus of ligand H₄A also the planar chelate [Ni(HA)₂]^4? with the donor set N₄ can be detected. The transition from [Ni(HA)]^? to [Ni(HA)₂]^4? is connected with a change of the coordination sphere (imino diacetic acid group → dioxime group) and the cis-trans isomerization of at least one oxime group. Protonation and stability constants are given and the properties of solid complexes are described.     

Comparison of the effects of different antiviral treatments on the antioxidant systems of stroma-free hemoglobin

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by > 3.69 log10 under conditions that inactivated 3.33 log10 of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log10 (1.37 J/cm2 irradiation and 2 microM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log10 (1.37 J/cm2 irradiation and 24 microM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log10 (60 degrees C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.     

Characterization of adsorption of humic acid onto alumina using quartz crystal microbalance with dissipation

In this paper, a quartz crystal microbalance with dissipation monitoring (QCM-D) is used to investigate humic acid (HA) adsorption onto alumina (Al(2)O(3)). The amount of adsorption and layer structures of HA were determined by the real-time monitoring of resonance frequency and energy dissipation changes (Δf and ΔD). The effect of HA concentration, HA molecular characteristics (molecular weight and polarity), and pH on HA adsorption onto Al(2)O(3) were investigated. The mass of HA adsorption increases as the concentration of HA increases. The masses are about 24, 60, and 87 ng cm(-2) as the concentration of DOC is 1.0, 4.85, and 92.0 mg L(-1), respectively. The adsorbed layer of HA is more nonrigid, and the mass of HA adsorption is higher at weakly acidic pH values. It was 20, 80, 65, and 45 ng cm(-2) at pH values of 4.5, 5.5, 6.5, and 8.0, respectively. This reveals that efficient HA removal by coagulation at weakly acidic pH values is not just due to the hydrolysis of Al ions as previously presumed. The adsorbed layer of hydrophobic HA is more nonrigid than hydrophobic HA (fractionated by Amberlite XAD-8 resin), and the mass adsorption for the hydrophobic fraction is about four times higher than the hydrophilic fraction (120 ng cm(-2) and 30 ng cm(-2)). The method is of value in the research to establish a quantified calculation model for the coagulation process.     

Extraction of humic acids and their fractions in poly(ethylene glycol)-based aqueous biphasic systems

The possibility of extraction and fractionation of humic acid (HA) in the aqueous biphasic systems (ABS) was shown for the first time. The 10% PEG-10% (NH₄)₂SO₄-H₂O and 5% PEG-7.5% dextran (or dextran sulphate, sodium salt)-H₂O systems were used. HA originated from peat, soddy-podzolic soil and chernozem were studied. The distribution coefficients were measured for HA of different origin, size of the molecules, molecular weights (MW) of the polymers and pH of the system. The PEG-(NH₄)₂SO₄-H₂O system was found to be better for preconcentration and isolation of HA, whereas the PEG-dextran-H₂O system is preferable for HA fractionation. The extractability of HA in ABS increases with increasing the MW of HA molecules. Peat HA are extracted in ABS with higher distribution coefficients compared with chernozem and soddy-podzolic soil HA. It is consistent with higher hydrophobicity of peat HA revealed by hydrophobic interaction chromatography. ABS are promising for HA separation into fractions that differ in their hydrophobic/hydrophilic properties as well as for comparing HA of different origin by their relative hydrophobicity.     

Synthesis and characterization of grafted nanohydroxyapatites using functionalized surface agents

Synthetic hydroxyapatite, HA [Ca10(PO4)6(OH)2], is a bioactive material that is chemically similar to biological apatite, the mineral phase of bone (a nanocomposite material). Synthetic biocomposites, comprising a polymer and hydroxyapatite that are used for bone replacement, have limitations when loaded under fatigue in that the weak mechanical bond between the two phases can result in failure at the interface. Chemical coupling of the HA and polymer matrix may provide a means of improving the interfacial bonding between the polymer and HA phases. Herein, we report our first steps toward developing chemically coupled nano-biocomposites via a two-step process. We describe the synthesis and characterization of surface-grafted hydroxyapatite (SG-HA), which possesses a reactive C=C functional group. In future work, we will report on the second step, namely the coupling of this functional group to a polymer by a copolymerization reaction to give a chemically coupled nano-biocomposite. The SG-HA reported herein was characterized by a range of methods including 31P and 13C magic-angle spinning (MAS)-NMR, Fourier transform infrared (FTIR), and Raman spectroscopy.     

蛋白质在羟基磷灰石界面吸附的研究

考察了酪蛋白酸钠(sodium caseinate,SC)和乳清分离蛋白(whey protein isolate,WPI)在表面性质不同的3种羟基磷灰石(hydroxyapatite,HA)颗粒上的界面吸附,分析了蛋白质的分子构型和HA颗粒的表面性质等因素对蛋白质在HA界面吸附的影响,重点讨论了SC和WPI肽链上磷酸化丝氨酸基团(phosphorylated serine residues,Ser-P)的数量和分布对吸附差异的影响.通过傅里叶变换红外光谱和表面电位分析发现SC和WPI无法被比表面积较小的HA颗粒有效吸附,但是在有效吸附面积较高的球状纳米HA和棒状微米HA上能够被吸附.Ser-P的存在使得SC在HA界面的吸附量更高、吸附能力更强.Ser-P数量和分布的不同则导致了SC中不同的蛋白组分在HA界面的竞争性吸附:β-酪蛋白在2μmHA界面始终存在优先吸附性;当纳米HA的浓度低于15 mg/mL时,纳米HA界面会优先吸附αs-酪蛋白.     

The basic research on physiological property of functionalized hyaluronan—I. Effect of hyaluronan and sulfated hyaluronan on cell proliferation of human epidermal keratinocytes

Hyaluronan (HA) is one of the polysaccharides that is found widely in connective tissue of mammals, and it has no sulfate group and high molecular weight in comparison with other glycosaminoglycans. Glycosaminoglycans are deeply concerned with the manifestation of biofunctions not only by their physical properties but also by physiological ones. In this study, sulfated HA (S‐HA) with various degrees of sulfate substitution and high molecular weight will be synthesized in order to give HA new biological functions. Moreover, the effect of HA and S‐HA on cell proliferation of human epidermal keratinocytes in vitro will be discussed. HA did not affect lag phase, but growth rate (metabolic turnover) of the cell in a logarithmic growth phase which was controlled by the molecular weight of HA. S‐HA stimulated the cell proliferation in the low concentration region under 1 μg/ml. While it inhibited the cell proliferation in the high concentration region over 10 μg/ml. It strongly suppressed the cell proliferation in the logarithmic growth metaphase. These facts were considered to be caused by the change of the cell‐matrix and/or cell–cell interactions. Copyright © 2004 John Wiley & Sons, Ltd.     

钛基多孔HA/SiO2复合涂层的制备及性能研究

为了制得表面多孔且与基材结合强度高的羟基磷灰石(HA)涂层,实验中以正丁醇为分散介质,以SiO2粉末为添加剂,纯钛片为基材,电泳沉积制备羟基磷灰石/二氧化硅/壳聚糖/(HA/SiO2/CS)复合涂层,经后续热处理得到多孔HA/SiO2复合涂层,采用扫描电镜(SEM)、傅立叶红外光谱仪(FT-IR)、X射线衍射仪(XRD)、万能材料试验机对涂层的表面形貌、组成、结构和结合强度进行测试和表征,并通过模拟体液(SBF)浸泡法对复合涂层的生物活性进行评价.结果表明:当悬浮液中的HA/SiO2/CS质量比为1∶1∶1时,制得的HA/SiO2/CS涂层经700℃热处理后获得的HA/SiO2复合涂层孔洞分布均匀,大孔孔径在10～15μm,小孔孔径在1～5μm;涂层与基材的结合强度达到25.5 MPa;多孔HA/SiO2复合涂层在SBF中浸泡7 d后,涂层表面碳磷灰石化;说明实验中添加SiO2所制得的多孔HA/SiO2复合涂层与钛基材结合强度高,且具有良好的生物活性.     

Two-dimensional packing patterns of amino acid surfactant and higher alcohols in an aqueous phase and their associated packing parameters

An amino acid surfactant, monosodium N-stearoyl-L-glutamate (MSSG), was assumed to associate with higher alcohols (HAs) in 1:1 molar ratio in an aqueous phase, and the packing parameters of the 1/1 MSSG/HA associates, Pa, were calculated by molecular dynamics (MD). Pa is defined as At1(l(t1)+l(t2))/a(h)l(t1), where At1 stands for the cross-sectional area of the MSSG tail, l(t1) for the length of the MSSG tail, l(t2) for the length of the HA tail, and a(h) for the area of the MSSG head group. The Pa value increased from 0.83 to 1.02 as the HA tail length (l(t2)) increased from C14 to C22. Associates of HAs having longer tails with MSSG are likely to fit better into bilayers because Pa is closer to 1 when l(t2) is longer. In the graphical results of the MD simulation for the association, however, a steric hindrance was found between the head groups of MSSG/HA when l(t2) was > or = C19. Based on this result, HA was classified into short-chain HA (SCHA, l(t2) < C19) and long-chain HA (LCHA, l(t2) > or = C19), and several possible packing units, composed of compositional combinations of MSSG, SCHA, and LCHA, were proposed. The packing unit is a building block which could constitute bilayers, and it is composed of variable compositional combinations of MSSG/HA. Assuming that SCHA associates with MSSG, the packing parameter, Punit, was calculated in a water box by MD for each packing unit. Punit is the packing parameter of a packing unit and it is defined as Vu/l(u)a(u), where Vu is the tail volume of the packing unit, l(u) is the chain length, and a(u) is the head area. For the calculation, stearyl alcohol (C18-OH, SA) was chosen as a SCHA and behenyl alcohol (C22-OH, BA) as a LCHA. When the compositional ratio MSSG:SCHA:LCHA was 1:1:1, Punit was around 1. The packing unit having Punit of around 1 formed a colloidally stable suspension for 30 days and its aggregate was a lamellar structure. However, the other packing units, for which Punit deviates significantly from 1, precipitated out in their suspensions and showed no evidence of a lamellar structure. According to the graphical MD simulations for the compositional MSSG/SCHA/LCHA associations in bilayers, vertical steric hindrance was found between LCHAs when Punit deviated significantly from 1. The steric hindrance would prevent the packing units from forming a stable bilayer and induce precipitation in the suspensions. Therefore, a proper combinational ratio of MSSG:LCHA:SCHA would play a major role in forming a lamellar structure.