纳米形态铝化合物与辅酶的作用和对相关脱氢酶活性的影响
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摘要
近年来,不同类型纳米粒子的安全性和毒性在世界范围内引起了广泛的关注。研究证实,纳米铝化合物可以改变有机物的性质,从而成为潜在的环境污染物,同时其生物毒性也引起了各方重视。纳米氧化物及氢氧化物可能是生理条件下环境和生物体系中铝的主要存在形态。本文通过探索合成了纳米铝化合物,并研究腺嘌呤二核苷酸(NAD+)及腺嘌呤二核苷酸磷酸(NADP+)在纳米铝化合物表面的吸附,探讨论了纳米铝化合物的吸附机理及其生物有效性。同时采用电化学方法研究了纳米形态及其他形态铝化合物对依赖于辅酶NAD(H)/NADP(H)的脱氢酶活性的影响,并利用荧光光谱和圆二色谱法研究了影响机制。主要内容如下：
1、合成了纳米-Al2O3,γ-Al2O3和AIOOH。研究了辅酶I(NAD+)在纳米γ-Al2O3、 α-Al2O3和γ-AlOOH的表面吸附,采用现代光谱分析手段UV-vis、ICP、XRD、TG-DTA、 XPS、ATR-FTIR和荧光光谱相结合进行分析。实验发现,NAD+的吸附量受pH值影响较大,pH值升高,吸附量迅速下降,且吸附作用明显受离子强度的影响,随着离子强度的增大,吸附量骤减。结果表明NAD+在纳米α-Al2O3和γ-AlOOH表面的吸附主要是外层吸附,而XPS和ATR-FTIR证明NAD+在γ-A12O3表面以外层吸附为主的同时存在少量内层配位方式。荧光实验证明,在研究范围内,随着纳米铝化合物用量的增大,改变了NAD+折叠式与展开式的构象平衡。
2、采用现代光谱分析手段研究了辅酶Ⅱ(NADP+)在纳米γ-Al2O3、α-Al2O3和γ-AlOOH表面的吸附行为。结果显示,NADP+的吸附量随pH值的升高而降低,3 3、概述了多壁碳纳米管和石墨烯修饰电极研究溶液中A1(III)和纳米A113对依赖于辅酶NAD(H)/NADP(H)的相关脱氢酶活性的影响。并采用滴涂法制备了还原型石墨烯CRG/CHIT修饰电极,利用计时电流i-t法,通过检测NADPH和NADH在电极上催化电流的变化情况,研究了纳米形态铝化合物(A12O3和AIOOH)对乙醇脱氢酶(ADH)和谷胱甘肽还原酶(GR)活性的影响。结果显示,CRG修饰电极能有效的降低NADPH和NADH在裸的玻碳电极上的氧化还原电位,纳米A12O3和AIOOH对GR和ADH的活性具有显著的抑制作用。通过Lineweaver Burk双倒数曲线计算了加入不同浓度纳米A12O3和AIOOH时相关脱氢酶的米氏常数,证明A12O3和AIOOH对GR和ADH的作用属于反竞争性抑制机制。同时荧光光谱法和圆二色谱法表明纳米A1203和AlOOH能够引起辅酶和脱氢酶本身的构象变化,从而引起酶活性的改变。实验证明,计时电流i-t法稳定、灵敏、快速有效,可以作为生物体系中酶活性测定的手段。
In recent years, the potential effects of nanoparticles, which cause unforeseen health or environmental hazards to human beings or other animal species, have attracted considerable concerns in the whole world. The interaction between nano-sized aluminum compounds and organics may change the properties of the organism, therefore, nano-aluminum compounds are considered as potential environmental pollutants. Under physiological conditions of biological systems, nano-sized oxides and hydroxides may be the exact species of aluminum. In this study, nano-sized aluminum compounds were synthesized and the adsorption of adenine dinucleotide (NAD+) and adenine dinucleotide phosphate (NADP+) on the synthesis nanoparticles were investigated. The experiments involved the adsorption mechanism and the impact on the biological systems. Meanwhile, electrochemical methods were performed to study the effect of different species of aluminum compounds on the activity of NAD(H)/NADP(H)-dependent dehydrogenases. The fluorescence and circular dichroism spectroscopy were applied to interpret the proposed mechanism. The main results can be concluded as follows:
1. Nano-sized α-Al2O3, γ-Al2O3and y-AlOOH were synthesized. And the adsorption behavior of NAD+on crystallized nano-sized γ-AlOOH,γ-Al2O3and α-Al2O3were investigated by using batch adsorption experiments and modern analytical methods. The methods included UV-vis, ICP, XRD, TG-DTA, XPS, ATR-FTIR and fluorescence spectroscopy over a range of different NAD+/NADP+concentrations, pH conditions and ionic strength conditions. The results showed that the adsorption capacity exhibited a strong dependence on pH values, that is, the amount of adsorbed coenzymes decreased as the pH values increased. Moreover, the facts indicated that the ligand adsorption also depended on the concentration of ionic-strength, which is the typical property of outer-sphere complex fashion. All observations suggested that NAD+on α-Al2O3and γ-AlOOH were predominantly adsorbed in outer-sphere mode. While in situ ATR-FTIR and XPS spectra of adsorbed NAD+indicated that there was some minor inner-sphere mode coupled with the outer-sphere coating on γ-Al2O3surface. Under the experimental conditions, the conformation of NAD+/NADP+changed with the addition of nanoparticles.
2. The adsorption behavior of NADP+on nano-sized γ-AlOOH, γ-Al2O3and α-Al2O3were studied by using modern analytical methods. The results showed that the adsorption capacity exhibited a strong dependence on pH values. As3 3. The research of the multi-wall carbon nanotube and graphene modified electrode were studied with the influence of Al(III) and Al13on the activity of the NAD(H)/NADP(H)-dependent dehydrogenases in solutions. The CRG/CHIT modified electrode was prepared and used to monitor the amperometric response of NADH and NADPH, which was applied to detect the activity of alcohol dehydrogenase (ADH) and glutathione reductase (GR). It was found that not only over potential of NADH/NADPH on the bare glassy carbon electrode was reduced, but also nano-sized Al2O3and AlOOH had been identically determined to display inhibition on activity of GR and ADH. According to Lineweaver Burk double reciprocal plot, the values of Michaelis constant Km for related dehydrogenases were calculated under different concentrations of nano-sized Al2O3and AlOOH added to the system. The properties of Km and Vmax indicated that the roles of nano-sized Al2O3and AlOOH in the ADH and GR catalytical system were ascribed as anti-competitive inhibitors. Moreover, fluorescence spectroscopy and circular dichroism (CD) spectra were applied to reveal the fact that nano-sized Al2O3and AlOOH could induce the conformational changes of both coenzymes and the related dehydrogenases, and then the unfavorable structural changes of substrate and enzyme would induce the change of enzyme activity. In a word, the results proved that the amperometric i-t curve could be applied as an effective tool to monitor the enzyme activity in the biological system due to its perfect property of stability, sensitivity and easy operation.
1、合成了纳米-Al2O3,γ-Al2O3和AIOOH。研究了辅酶I(NAD+)在纳米γ-Al2O3、 α-Al2O3和γ-AlOOH的表面吸附,采用现代光谱分析手段UV-vis、ICP、XRD、TG-DTA、 XPS、ATR-FTIR和荧光光谱相结合进行分析。实验发现,NAD+的吸附量受pH值影响较大,pH值升高,吸附量迅速下降,且吸附作用明显受离子强度的影响,随着离子强度的增大,吸附量骤减。结果表明NAD+在纳米α-Al2O3和γ-AlOOH表面的吸附主要是外层吸附,而XPS和ATR-FTIR证明NAD+在γ-A12O3表面以外层吸附为主的同时存在少量内层配位方式。荧光实验证明,在研究范围内,随着纳米铝化合物用量的增大,改变了NAD+折叠式与展开式的构象平衡。
2、采用现代光谱分析手段研究了辅酶Ⅱ(NADP+)在纳米γ-Al2O3、α-Al2O3和γ-AlOOH表面的吸附行为。结果显示,NADP+的吸附量随pH值的升高而降低,3
In recent years, the potential effects of nanoparticles, which cause unforeseen health or environmental hazards to human beings or other animal species, have attracted considerable concerns in the whole world. The interaction between nano-sized aluminum compounds and organics may change the properties of the organism, therefore, nano-aluminum compounds are considered as potential environmental pollutants. Under physiological conditions of biological systems, nano-sized oxides and hydroxides may be the exact species of aluminum. In this study, nano-sized aluminum compounds were synthesized and the adsorption of adenine dinucleotide (NAD+) and adenine dinucleotide phosphate (NADP+) on the synthesis nanoparticles were investigated. The experiments involved the adsorption mechanism and the impact on the biological systems. Meanwhile, electrochemical methods were performed to study the effect of different species of aluminum compounds on the activity of NAD(H)/NADP(H)-dependent dehydrogenases. The fluorescence and circular dichroism spectroscopy were applied to interpret the proposed mechanism. The main results can be concluded as follows:
1. Nano-sized α-Al2O3, γ-Al2O3and y-AlOOH were synthesized. And the adsorption behavior of NAD+on crystallized nano-sized γ-AlOOH,γ-Al2O3and α-Al2O3were investigated by using batch adsorption experiments and modern analytical methods. The methods included UV-vis, ICP, XRD, TG-DTA, XPS, ATR-FTIR and fluorescence spectroscopy over a range of different NAD+/NADP+concentrations, pH conditions and ionic strength conditions. The results showed that the adsorption capacity exhibited a strong dependence on pH values, that is, the amount of adsorbed coenzymes decreased as the pH values increased. Moreover, the facts indicated that the ligand adsorption also depended on the concentration of ionic-strength, which is the typical property of outer-sphere complex fashion. All observations suggested that NAD+on α-Al2O3and γ-AlOOH were predominantly adsorbed in outer-sphere mode. While in situ ATR-FTIR and XPS spectra of adsorbed NAD+indicated that there was some minor inner-sphere mode coupled with the outer-sphere coating on γ-Al2O3surface. Under the experimental conditions, the conformation of NAD+/NADP+changed with the addition of nanoparticles.
2. The adsorption behavior of NADP+on nano-sized γ-AlOOH, γ-Al2O3and α-Al2O3were studied by using modern analytical methods. The results showed that the adsorption capacity exhibited a strong dependence on pH values. As3
引文
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