乙炔基自由基C2H与氧气反应的密度泛函理论研究

应用量子化学从头算和密度泛函理论(DFT)对C₂H自由基和O₂的反应进行了研究.在B3LYP/6-311G^**水平上优化了反应通道上各驻点(反应物、中间体、过渡态和产物)的几何构型,并计算出它们的振动频率和零点振动能(ZPVE).各物种的总能量由CCSD(T)/6-311G^**//B3LYP/6-311G^**给出,并对能量进行了零点能校正.计算结果表明,反应物中自由基C₂H中的边端C进攻O₂形成了中间体1 (HCCOO),中间体1是一个加合产物.由中间体1经过不同的反应通道可以生成不同的产物P₁ (HCO+CO), P₂ (HCCO+O), P₃(CO₂+CH), P₄ (C₂O+OH)和P₅ (2CO+H).反应通道之间存在着竞争机制.其中P₁, P₂是主要产物,其次还有一定比例的P5生成,而产物P₃, P₄的生成几率较低.各条反应通道化学反应热的计算与实验吻合较好.     

镧系元素Dawson结构钨钒磷四元杂多配合物的合成、表征以及对H2O2分解的催化活性

合成了通式为K₁₅H₃[Ce(P₂W₁₆VO₆₁)₂]·61H₂O、K₁₅H₄[Ln(P₂W₁₆VO₆₁)₂]·xH₂O(Ln=La³⁺,Pr³⁺,Nd³⁺,Sm³⁺,Eu³⁺,Gd³⁺,Dy³⁺,Yb³⁺)的9种镧系元素Dawson结构的钨钒磷四元杂多配合物,并用IR、UV、NMR、ESR、XRD、TG-DTA等对其结构和性质进行了研究。该类配合物具有与K₁₆[Ce(P₂W₁₇VO₆₁)₂]·50H₂O类似的结构,对H₂O₂分解有较高的催化活性。     

LiNd(PO3)4晶体的热分析研究

对LiNd(PO₃)₄晶体分别在N₂气和空气下进行了TG和DTA热分析研究,给出TG和DTA曲线,讨论了LNP晶体在N₂气和空气下不同的热分解机理.得到分解产物分别为Nd₄(P₂O₇)₃和NdP₂O₇.     

H6[P2Mo18O62]的钒取代多金属氧酸盐对小鼠黑色素瘤细胞B16黑色素生成的调控及其机制研究

合成了五种多金属氧酸盐. 以B16小鼠黑色素瘤细胞为模型, 用噻唑蓝(MTT)法检测细胞存活率, 酶标法测定抗氧化活性和内黑色素含量及酪氨酸酶活性, 最后用分子对接模拟多金属氧酸盐和酪氨酸酶结合的机制. 研究结果表明, 两种磷钼酸(H₇[P₂Mo₁₇VO₆₂]和H₈[P₂Mo₁₆V₂O₆₂])是高效的黑色素生成抑制剂, 在200 μmol/L的浓度下对黑色素合成的抑制率为74.40%和75.14%, 对细胞内酪氨酸酶活性的抑制率为35.71%和40.00%, 随着钒原子取代个数的增加, 两种抑制活性均逐渐降低. H₇[P₂Mo₁₇VO₆₂]和H₈[P₂Mo₁₆V₂O₆₂]对细胞没有毒性. 两种多酸都有较好的1,1-二苯基-2-三硝基苯肼(DPPH)清除能力, IC₅₀分别为1.683和2.800 mg/mL. 分子对接分数低于–146 kJ/mol. 综上所述, H₇[P₂Mo₁₇VO₆₂]和H₈[P₂Mo₁₆V₂O₆₂]能抑制B16细胞黑色素的生成, 其机制与抑制酪氨酸酶的活性有关.     

具有Dawson结构的钼钒磷杂多酸氧化还原性质的研究

通过SnCl₂电位滴定、极谱和循环伏安方法,详细研究了2:18系列钼钒磷杂多酸H_(6+n)[P₂Mo^13-nV_nO₆₂]·xH₂O(n=1～3)和H^_4+n[P₂Mo_18-nV_nO₆₂H₂]·xH₂O(n=4,5)在溶液中的氧化还原性质,证实它们的还原为三步分别加合n,2,2个电子,同时加合n,2,2个质子的过程,总结了循环伏安峰电位与pH的关系,提出了还原机理。     

ClONO2与Cl(2P3/2)的反应机理

在密度泛函理论B3LYP/6-31G^*水平上,研究了ClONO₂+Cl(²P_3/2)Cl₂+NO₃和ClONO₂+Cl(²P_3/2)ClO+ClONO(cis)及ClONO₂+Cl(²P_3/2)ClOCl+NO₂的反应机理.计算得到各可能反应途径的过渡态,并经过内禀反应坐标(IRC)分析加以证实.反应ClONO₂+Cl(²P_3/2)Cl₂+NO₃反应活化能垒最低,为4.5kJ/mol,是反应主通道.     

MnSAPO-34分子筛的制备、表征及其NH3-SCR活性

采用水热合成方法制备含锰的SAPO-34分子筛(MnSAPO-34)催化剂,考察了锰投加量、焙烧温度及晶化时间对催化剂氨选择性催化还原(SCR)氮氧化物反应活性的影响,并通过X射线光电子能谱(XPS)、程序升温还原(TPR)、程序升温脱附(TPD)等多种分析手段对催化剂进行表征.活性测试结果表明,当MnO与P₂O₅的摩尔比n_(MnO)/n_(P2O5)= 0.1,采用6 h晶化时间, 550 ℃焙烧制备的MnSAPO-34分子筛具有最佳SCR活性, NO_x转化率接近100%, N₂选择性高于80%.分析结果表明, Mn的引入对分子筛的晶体及多孔结构有较大影响,过多的引入会降低结晶度及产生非骨架锰氧化物,同时还会降低分子筛的比表面积和孔容,但焙烧温度的降低以及晶化时间的缩短可以提高分子筛的比表面积和孔容.高温焙烧后分子筛表面出现了高氧化态锰物种,以Mn⁴⁺为主,而提高Mn³⁺的比例则有利于提高催化活性.在适当的合成条件下, Mn的引入可增强分子筛对NO和NH₃分子的吸附,而强吸附态NO及强吸附态NH₃的相互作用可能是催化活性快速提高的原因.     

Mn/Ce-ZrO2催化剂低温NH3-SCR脱硝性能研究

采用浸渍法制备了三种具有不同载体的锰基NH₃低温选择性催化还原(NH₃-SCR)催化剂Mn/Ce-ZrO₂、Mn/P₂₅和Mn/Al₂O₃。研究了三种催化剂低温SCR脱硝活性及抗H₂O、抗SO₂性能,并采用XRD、NH₃-TPD和H₂-TPR手段对催化剂的物理化学性质进行表征。结果表明,在无H₂O和SO₂存在的情况下,三种催化剂的低温SCR催化活性均比较高。相对来说,Mn/Ce-ZrO₂在低温段(100~160℃)活性更高,Mn/P₂₅在高温段(160~220℃)活性更高,这与两种催化剂的氧化还原性质有关。H₂-TPR表征表明,Mn/Ce-ZrO₂更容易发生氧化还原反应,而Mn/P₂₅还原峰对应的温度较高、面积较大。三种催化剂均有很高的低温抗水性能。另外,Mn/Ce-ZrO₂的抗H₂O、抗SO₂性能最好,而Mn/P₂₅的抗H₂O、抗SO₂性能最差。Mn/Ce-ZrO₂具有较好的抗H₂O、抗SO₂性能是由于其具有较多的表面酸位点,且表面生成的硫铵盐不稳定。     

食品中苏丹红Ⅰ和辣椒红的表面增强拉曼散射(SERS)光谱与激发-发射矩阵荧光光谱鉴别

利用表面增强拉曼散射(SERS)光谱及激发-发射矩阵(EEM)荧光光谱对食品中非法添加剂苏丹红Ⅰ和合法食品添加剂辣椒红进行了定性分析和检测. SERS光谱结果表明, 苏丹红Ⅰ在低波数区域分子的扭转振动信号增强比较明显; 而辣椒红在1521和1158 cm^-1处拉曼信号增强效果比较明显; EEM荧光光谱结果表明, 苏丹红Ⅰ的乙醇溶液在P₁(λ_ex=285 nm, λ_em=345 nm)和P₂(λ_ex= 335 nm, λ_em=548 nm)处有2个明显的荧光特征峰; 而辣椒红的乙醇溶液有3个特征荧光峰, 分别为P₁(λ_ex=545 nm, λ_em=580 nm), P₂(λ_ex=560 nm, λ_em=665 nm)和P₃(λ_ex=608 nm, λ_em=672 nm).     

钒取代的Dawson型磷钼酸对酪氨酸酶的抑制作用

采用酶动力学方法研究了5种钒取代的Dawson型磷钼酸H₇[P₂Mo₁₇VO₆₂]、H₈[P₂Mo₁₆V₂O₆₂]、H₉[P₂Mo₁₅V₃O₆₂]、H₈[P₂Mo₁₄V₄O₆₂H₂]和H₉[P₂Mo₁₃V₅O₆₂H₂](分别简写为P₂Mo₁₇V、P₂Mo₁₆V₂、P₂Mo₁₅V₃、P₂Mo₁₄V₄和P₂Mo₁₃V₅)对蘑菇酪氨酸酶二酚酶的抑制作用,结果表明,效应物P₂Mo₁₇V、P₂Mo₁₆V₂和P₂Mo₁₅V₃能够明显地抑制酪氨酸酶的活性,其半抑制浓度(IC₅₀)值分别为0.409、0.386和0.386 mmol/L,且均表现为可逆的竞争型抑制,效应物P₂Mo₁₇V、P₂Mo₁₆V₂和P₂Mo₁₅V₃对游离酶的抑制常数K_I分别为0.234、0.391和0.249 mmol/L。 而效应物P₂Mo₁₄V₄在0~1.0 mmol/L浓度范围内,对酪氨酸酶二酚酶无明显抑制作用,效应物P₂Mo₁₃V₅对酪氨酸酶二酚酶表现为激活作用。     

Proteomics Approach to Investigate Impact of aFGF Antagonist on Signal Transduction Pathway

Fibroblast growth factor(FGF) signal transduction pathway is correlated with many diseases,such as cancer,skeletal dysplasias,olfactory syndromes and so on.P(VYMSPF),a small peptide,is an antagonist of aFGF.In order to investigate the impact of the peptide P on aFGF signal transduction pathway,the proteomes of NIH3T3 cell stimulated under different conditions(aFGF+heparin sodium and aFGF+heparin sodium+P) were extracted and analyzed by 2DE,five differentially expressed protein spots were selected and identified via ESI-MS/MS.The result indicates that the peptide P has a great influence on the function of aFGF such as metabolism,protein translation and cytoskeleton formation;the target signal pathway of the peptide P may be MAPK pathway and the peptide P might also affect the PLCγ pathway.The article provides a new insight into the peptide P as a potential anticancer drug.     

Matrix assisted laser desorption/ionization-time of flight-mass spectrometry analysis of proteins detected by anti-phosphotyrosine antibody on two-dimensional-gels of fibrolast cell lysates after tumor necrosis factor-alpha stimulation

We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-alpha. After stimulation with TNF-alpha, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10-30 min time intervals, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immunoblot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hundred protein spots were detected in the TNF-alpha stimulated L929 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel corresponding to those detected by immunoblot analysis were subjected to in-gel trypsin digestion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysis were identified by MS-Fit database search analysis. Among them, the proteins that show time-dependent changes in staining intensity include vimentin, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-alpha. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of time-dependent changes in many proteins that are involved in signal transduction. Usefulness of the method for comprehensive analysis of the proteins involved in signal transduction pathway and the limitations of the method are discussed.     

Analysis of Peptide Ligand Binding to FGFR1

Simulating annealing algorithm was used in docking computation to predict a selected peptide VYMSPF(P2) binding site on the ectodomain of FGFR1.The peptide is located on the hydrophobic surface of the receptor,which is critical for FGF binding.The synthesized peptide can effectively inhibit the mitogenic activity of afGF,and has a potential to become a therapeutic agent as an aFGF antagonist.     

Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry

The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin-enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two-dimensional (2-D) maps and subsequent identification of over 100 protein spots by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post-translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold-detergent method yielded a large number of known caveolae residents, including caveolin-1. Thus, a large part of this subproteome was established, including known (trans-)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)-anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP-2, C8ORF2, and XRP-2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.     

质谱及相关技术在信号传导方面的研究进展

细胞信号传导是近年来生命科学研究的热点之一。有关蛋白转录后修饰 (如蛋白质磷酸化、乙酰化、糖基化等 ) ,信号肽序列测定 ,信号传导途径和多通道调节方式 ,蛋白自折叠及构象变化 ,小分子脂类信号分子等研究由于质谱技术的快速发展而取得了突破性的进展     

Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.     

嗅觉记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了C57BL/6J小鼠在嗅觉训练和记忆测试后嗅球蛋白表达的差异, 探讨了与嗅觉记忆相关的蛋白质. C57BL/6J小鼠经嗅觉训练后, 可对相应的气味保持记忆能力, 其嗅球蛋白表达存在明显差异, 5种蛋白与嗅觉记忆形成显著相关. 上述蛋白功能涉及神经发育, 信号转导及细胞骨架和核苷酸代谢, 其中神经发育和信号转导相关蛋白表达上调, 而细胞骨架和核苷酸代谢相关蛋白表达水平降低. 这些与嗅觉记忆形成相关的蛋白深化了对嗅觉记忆机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Sensitive and versatile electrogenerated chemiluminescence biosensing platform for protein kinase based on Ru(bpy)3 functionalized gold nanoparticles mediated signal transduction

A novel, sensitive and versatile electrogenerated chemiluminescence biosensing platform is developed for monitoring activity and inhibition of protein kinase based on Ru(bpy)₃²⁺ functionalized gold nanoparticles (Ru(bpy)₃²⁺-AuNPs) mediated signal transduction. Ru(bpy)₃²⁺-AuNPs were formed by functionalizing AuNPs with Ru(bpy)₃²⁺ through electrostatic interactions and were used as thiol-versatile signal probe. Casein kinase II (CK2) and cAMP-dependent protein kinase (PKA), two classical protein kinase implicated in disease, were chosen as model protein kinases while a CK2-specific peptide (CRRRADDSDDDDD) and a PKA-specific peptide (CLRRASLG) were employed as molecular substrate for CK2 and PKA, respectively. The specific peptide was self-assembled onto the gold electrode via Au–S bond to form ECL biosensor. Upon thiophosphorylation of the peptide on the electrode in the presence of protein kinase and co-substrate adenosine-5’-(γ-thio)-triphosphate, Ru(bpy)₃²⁺-AuNPs was assembled onto the thiophosphorylated peptides via Au–S bond. The Ru(bpy)₃²⁺-AuNPs attached on electrode surface produce detectable ECL signal in the presence of coreactant tripropylamine. This strategy is promising for multiple protein kinase assay and kinase inhibitor profiling with high sensitivity, good selectivity and versatility. The ECL intensity is proportional to the activity of CK2 in the range of 0.01–0.5 unit/mL with a low detection limit of 0.008 unit/mL and to the activity of PKA in the range of 0.01–0.4 unit/mL with a detection limit of 0.005 unit/mL. Additionally, this assay was applied to the detection of CK2 in serum samples and the inhibition of CK2 and PKA. This work demonstrates that the developed ECL method can provide a sensitive and versatile platform for the detection of kinase activity and drug-screening.