生物无机化学在生活中的应用——金属蛋白质和金属酶

生物无机化学是研究生命体系中金属离子及其配合物存在状态和功能的科学,是配位化学和生命科学交叉的前沿学科。金属离子在生命体系中主要是通过金属离子与体内的酶、蛋白或核酸的相互作用以及存在体内的金属蛋白、金属酶行使其功能,因此通过现代物理方法来研究它们的结构、构象以及结构与功能的关系十分重要。     

温度和时间对肌红蛋白血红素铁与金属离子相互作用的影响

目的:研究在生理条件下,温度和时间对肌红蛋白血红素铁与各种金属离子直接相互作用的影响。方法:利用紫外-可见光谱法研究肌红蛋白活性中心的铁和外加金属离子(CuSO4、ZnSO4、MgSO4、CoCl2和MnSO4)的直接相互作用;改变作用温度(4、21、37和52℃)和作用时间(2、4、6、8和10 d),研究肌红蛋白活性中心铁卟啉与不同金属离子的直接相互作用。结果与结论:紫外光谱数据表明,金属离子与肌红蛋白活性中心的Fe(Ⅱ)发生直接相互作用,且随着作用温度的升高和作用时间的延长,这种相互作用逐渐增强。作用10 d后,金属离子与肌红蛋白活性中心的铁的作用强度依次为Mn(Ⅱ)>Zn(Ⅱ)>Co(Ⅱ);温度升至52℃时,作用强度依次为Cu(Ⅱ)>Mg(Ⅱ)>Zn(Ⅱ)。     

人体胆结石中的周期性环状结构及其形成机理研究

在一简化了的化学模拟体系—凝胶介质中,首先对胆结石中存在的二价铜、钙、镁及三价铁等主要金属离子各自的作用分别进行了研究。发现凝胶介质中金属离子和胆酸（根）的相互作用与离子种类有关：有的形成了球形颗粒沉淀;有的形成了周期性的沉淀带（环）;还有的在形成周期性沉淀的同时,产生了分形结构。在多离子体系中,发现不同离子之间既相互作用,相互协作又相对独立。表明了金属离子和胆酸之间的相互作用与胆结石中的环状结构有密切的联系,为深入研究胆结石中环结构的形成提供了重要的实验证据和线索。     

肼类与蓝膜的相互作用

采用紫外可见光谱、动力学光谱和荧光光谱,研究了肼、甲基肼和偏二甲基肼与蓝膜的相互作用。结果表明这些肼类都可使蓝膜恢复为紫膜,并恢复紫膜原有的光循环方式。但是,中间体(M412)的衰減速率加快,这在金属离子重组紫膜中没有发生。另外,当肼加入蓝膜时,荧光光谱没有明显变化;而去掉金属离子的蓝膜,其荧光強度明显增加。     

Zn~(2+)对EGCG的结构和抑菌活性的影响

揭示耻垢分枝杆菌(Mycobacterium smegmatis mc~2155)和Zn~(2+)对表没食子儿茶素没食子酸酯(Epigallocatechin-3-gallate,EGCG)结构和生物学活性的影响,为开发高活性EGCG制剂奠定基础。首先利用HPLC检测EGCG和耻垢分枝杆菌相互作用后的含量和结构变化,进而化学合成EGCG-Zn~(2+),利用紫外吸收光谱法和HPLC方法鉴定EGCG被Zn~(2+)修饰前后的结构变化。最后采用纸片琼脂扩散法比较EGCG和EGCG-Zn~(2+)对耻垢分枝杆菌的抑制作用。EGCG与耻垢分枝杆菌相互作用18 h后,即出现不同的EGCG代谢物,且EGCG自身结构含量比例明显降低。EGCG与特定金属离子Zn~(2+)结合后,其最大吸收波长和吸收强度均有改变,且EGCG-Zn~(2+)在紫外吸收光区有明显的酚羟基吸收。HPLC结果表明, EGCG-Zn~(2+)能引起EGCG结构变化而导致保留时间延长。抑菌实验研究发现,EGCG-Zn~(2+)对耻垢分枝杆菌的抑菌作用弱于EGCG。研究表明,EGCG结构的稳定性容易受到细菌以及Zn~(2+)的影响,为EGCG的进一步研究开发提供线索。     

光源或培养基成分对金花茶愈伤组织中DFR、LAR与PPO基因表达及总儿茶素含量的影响

该研究以富含儿茶素的金花茶愈伤组织为材料,对不同光源、激素、碳源及苯丙氨酸处理30 d的愈伤组织中DFR表达量、LAR表达量、PPO表达量与总儿茶素含量的变化情况及四者两两之间的相关性进行了分析.结果表明:这4个检测项目均对以上处理有显著的响应;在以上各因素处理下,DFR与LAR的表达模式十分相似,其相关系数处在0.710~0.889之间;在不同碳源处理下,PPO表达量与总儿茶素含量的变化呈显著负相关关系,其相关系数为-0.696;在不同苯丙氨酸添加量处理下,DFR与LAR表达量变化均与总儿茶素含量变化呈显著正相关关系,其相关系数分别为0.786和0.564;适宜儿茶素离体生产的金花茶愈伤组织增殖配方为附加4 mg·L-16-BA、0.6 mg·L-12,4-D、30 g·L-1蔗糖与0.6608 g·L-1苯丙氨酸的MS固体培养基,其总儿茶素含量可达40.11 mg·g-1 DW.以上研究表明,与茶树相似,在金花茶中DFR与LAR在儿茶素代谢过程中密切相关;PPO表达量升高导致金花茶儿茶素损失;添加适宜浓度的苯丙氨酸作为前体物质是提高愈伤组织中总儿茶素含量的有效措施.     

水环境中腐殖质-金属离子键合作用研究进展

腐殖质 (主要指腐殖酸和富里酸 )普遍存在于各种水体中 ,它对金属离子的形态、迁移转化、生物可利用性等地球化学行为起着重要作用。本文概述了水环境中腐殖质的一些基本性质 ,以及腐殖质 金属离子之间的键合作用机理、研究方法和影响因素。并且对各种金属离子键合到腐殖质上的现代物理化学模型 ,尤其对ModelⅥ及NICA Donnan模型进行了简要回顾和评述。它们在许多条件下模拟腐殖质 -金属离子键合作用可以得到令人欣喜的结果。还简述了腐殖质对水环境中金属离子各种水环境地球化学行为的影响。但是 ,若要更深入了解和阐述金属离子在水环境中的各种行为 ,还需考虑腐殖质与颗粒物质、胶体物质以及微生物等的相互作用。     

利用扰动角关联方法研究络合物中钼的氧化态及其配位体

扰动角关联方法是一种超精细相互作用的核技术,用于测定生物大分子在溶液中的旋转关联时间,也可用来研究溶液中生物大分子构型的变化、金属离子的介离常数及金属离子在生物体内的化学状态等等。本文利用~(99)Mo作为扰动角关联测量的探针核,以不同的价态分别与EDTA,酒石酸、半胱氨酸等络合。通过不同的扰动角关联衰减因子,分别研究了~(99)Mo周围不同的电场梯度,为金属离子在化合物中的价态和配位结构的微观研究开辟了一条新的途径。     

中性红再生紫膜的光化学性质

研究了中性红再生紫膜从先适应状态到暗适应状态的反应及再生紫膜中中性红的光吸收变化。实验结果说明紫膜上的金属离子结合位点可能深入膜内的质子通道,与构成质子通道的一些重要氨基酸残基相互作用。紫膜经去离子化处理变成蓝膜后,带有正电荷的质子化中性红也可以占据此金属离子结合位点,使蓝膜再生为紫膜。但金属离子与结合位点具有更强的亲和力,使蓝膜再生为紫膜的能力比质子化中性红强。     

茶儿茶素抑制肿瘤血管生成的作用

对近几年茶儿茶素抑制肿瘤血管生成的研究进展进行了综述。从抑制血管内皮细胞生长、抑制细胞黏附分子表达和抑制基底膜降解三个部分讨论了其抗肿瘤血管生成的机制,并对儿茶素研究存在的问题和儿茶素开发前景进行了探讨。     

Effects of pH and metal ions on antioxidative activities of catechins

The Effects of pH on antioxidative activities of catechol, pyrogallol, and four catechins, and effects of metal ions (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, and Zn2+) on antioxidative activities of (-)-epigallocatechin gallate (EGCG) were studied by an oxygen electrode method. The antioxidative activities of catechins were high and constant at pH 6-12, but decreased in acidic and strong alkaline solutions. Copper(II) ion the most strongly increased the antioxidative activity of EGCG among these metal ions examined, but iron(II) ion largely inhibited the antioxidative activity of EGCG. These effects are discussed considering the formation of metal complexes with catechins and the change in oxidation potentials.     

DNA cleavage activities of (-)-epigallocatechin, (-)-epicatechin, (+)-catechin, and (-)-epigallocatechin gallate with various kinds of metal ions

The DNA cleavage activities of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCg) were examined with 16 different metal ions. Cu(2+) with all the catechins facilitated DNA cleavage, while Ag+ with EGC and EC showed a strong repressive effect. The other metal ions examined showed little effect.     

Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.     

Functional identification of ligands for a catalytic metal ion in group I introns

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.     

Interaction of lactic acid bacteria with metal ions: opportunities for improving food safety and quality

Certain species of lactic acid bacteria (LAB), as well as other microorganisms, can bind metal ions to their cells surface or transport and store them inside the cell. Due to this fact, over the past few years interactions of metal ions with LAB have been intensively investigated in order to develop the usage of these bacteria in new biotechnology processes in addition to their health and probiotic aspects. Preliminary studies in model aqueous solutions yielded LAB with high absorption potential for toxic and essential metal ions, which can be used for improving food safety and quality. This paper provides an overview of results obtained by LAB application in toxic metal ions removing from drinking water, food and human body, as well as production of functional foods and nutraceutics. The biosorption abilities of LAB towards metal ions are emphasized. The binding mechanisms, as well as the parameters influencing the passive and active uptake are analyzed.     

Functional identification of catalytic metal ion binding sites within RNA

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.     

Interchangeable utilization of metals: New perspectives on the impacts of metal ions employed in ancient and extant biomolecules

Metal ions provide considerable functionality across biological systems, and their utilization within biomolecules has adapted through changes in the chemical environment to maintain the activity they facilitate. While ancient earth''s atmosphere was rich in iron and manganese and low in oxygen, periods of atmospheric oxygenation significantly altered the availability of certain metal ions, resulting in ion replacement within biomolecules. This adaptation mechanism has given rise to the phenomenon of metal cofactor interchangeability, whereby contemporary proteins and nucleic acids interact with multiple metal ions interchangeably, with different coordinated metals influencing biological activity, stability, and toxic potential. The ability of extant organisms to adapt to fluctuating metal availability remains relevant in a number of crucial biomolecules, including the superoxide dismutases of the antioxidant defense systems and ribonucleotide reductases. These well-studied and ancient enzymes illustrate the potential for metal interchangeability and adaptive utilization. More recently, the ribosome has also been demonstrated to exhibit interchangeable interactions with metal ions with impacts on function, stability, and stress adaptation. Using these and other examples, here we review the biological significance of interchangeable metal ions from a new angle that combines both biochemical and evolutionary viewpoints. The geochemical pressures and chemical properties that underlie biological metal utilization are discussed in the context of their impact on modern disease states and treatments.     

Prediction of transition metal-binding sites from apo protein structures

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.     

Determination of catechins and catechin gallates in tissues by liquid chromatography with coulometric array detection and selective solid phase extraction

Catechins levels in organ tissues, particularly liver, determined by published methods are unexpectedly low, probably due to the release of oxidative enzymes, metal ions and reactive metabolites from tissue cells during homogenization and to the pro-oxidant effects of ascorbic acid during sample processing in the presence of metal ions. We describe a new method for simultaneous analysis of eight catechins in tissue: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG) (Fig. 1). The new extraction procedure utilized a methanol/ethylacetate/dithionite (2:1:3) mixture during homogenization for simultaneous enzyme precipitation and antioxidant protection. Selective solid phase extraction was used to remove most interfering bio-matrices. Reversed phase HPLC with CoulArray detection was used to determine the eight catechins simultaneously within 25 min. Good linearity (>0.9922) was obtained in the range 20-4000 ng/g. The coefficients of variance (CV) were less than 5%. Absolute recovery ranged from 62 to 96%, accuracy 92.5 +/- 4.5 to 104.9 +/- 6%. The detection limit was 5 ng/g. This method is capable for determining catechins in rat tissues of liver, brain, spleen, and kidney. The method is robust, reproducible, with high recovery, and has been validated for both in vitro and in vivo sample analysis.