ESI质谱法对黄酮-7-磷酰化氨基酸酯与溶菌酶相互作用的研究

采用电喷雾(ESI)质谱技术,研究了4种黄酮-7-磷酰化氨基酸酯与溶菌酶的弱相互作用,实验结果表明4种化合物均能与溶菌酶形成非共价复合物,在相同条件下,未检测到黄酮与溶菌酶形成的非共价复合物,说明在黄酮分子中引入N-磷酰化氨基酸,能改变黄酮的分子极性,从而使其与生物大分子之间的相互作用情况发生变化;通过改变锥孔电压,分别对4种黄酮-7-磷酰化氨基酸酯-溶菌酶复合物的耐压能力进行检测.结果显示,黄酮-7-磷酰化氨基酸酯b与溶菌酶形成的复合物最稳定,它们之间存在最强的弱相互作用.     

荧光及ESI质谱法研究溶菌酶与磷酰化黄酮的相互作用

分别用荧光法和ESI质谱法研究了磷酰化黄酮和溶菌酶的相互作用 .结果均显示磷酰化黄酮能够和溶菌酶发生弱相互作用 ,与黄酮相比它对溶菌酶更具亲和力 .根据荧光猝灭双倒数图计算了磷酰化黄酮与溶菌酶之间的结合常数为k2 0℃ =1.68× 10 4L/mol,k3 7℃ =1.0 6× 10 4L/mol,实验证明随着温度的升高 ,磷酰化黄酮与溶菌酶的结合常数逐渐降低 ,说明了两者之间形成了复合物 ,此荧光猝灭过程为静态猝灭 .根据F ster能量传递原理计算出磷酰化黄酮在溶菌酶上的结合距离 ,并根据热力学参数确定了磷酰化黄酮与溶菌酶之间的作用力类型为电荷作用力     

大豆苷元磷酰化衍生物的合成及其与溶菌酶复合物的ESI研究

通过改进的Atherton-Todd反应，得到了5个新的大豆苷元磷酰化衍生物，其结构被X-ray,IR, NMR和ESI确认. ESI质谱证实在气态下磷酰化大豆苷元能和溶菌酶形成非共价复合物，而大豆苷元与溶菌酶的非共价复合物未能检测到. 通过变换ESI的orificevoltage，检测了不同的磷酰化大豆苷元与溶菌酶非共价复合物的相对稳定性.     

N-磷酰化肽酯及小肽与溶菌酶相互作用的ESI-MS研究

用ESI-MS研究了一系列结构具有可比性的N-磷酰化肽酯及小肽和溶菌酶的非共价相互作用, 比较了磷酰化肽酯及小肽分子中的不同基团对相互作用的影响. 结果表明—OH对其与溶菌酶的相互作用有较大贡献; 芳香环由于位阻原因, 对相互作用有促进和阻碍双重效应; 当—OH与芳香环相连时会发生协同效应, 可使相互作用显著增强. 磷酰化肽酯及小肽的体积大小、空间位阻对相互作用亦有显著影响. 磷酰化二肽中氨基酸残基的构型、顺序、碳链长短的变化(增加1～2个C)对其与蛋白溶菌酶之间的相互作用在质谱中没有表现出影响. 分子结构较为伸展、分子柔顺性好、空间位阻较小的磷酰化小肽更容易使蛋白在溶液中的构象趋于收缩, 而构象较为收缩的蛋白分子更易结合空间位阻较小的磷酰化小肽分子.     

磷酰化对丙氨酸与溶菌酶相互作用的影响

在电喷雾离子阱质谱图中发现丙氨酸不能和溶菌酶形成二聚体, 而磷酰化丙氨酸(DIPP-Ala)能和溶菌酶形成二聚体. 进一步研究发现丙氨酸及其他氨基酸磷酰化后, 自身形成二聚能力大大增强. 在Silicon Graplics图形工作站上采用SYBYL 6.8软件, 利用Tripos力场和分子力学方法研究了DIPP-Ala最低能量构象, 并用分子对接(DOCK)研究了二聚体的形成. 结果说明磷氧双键的存在增强了分子间的相互作用.     

N-磷酰化肽酯及小肽与氨基酸相互作用的ESI-MS研究

采用ESI-MS研究了一系列结构具有可比性的N-二异丙氧基磷酰化肽酯及小肽同20种蛋白氨基酸及3种D型氨基酸(D-Ala、D-Ser、D-Phe)的非共价相互作用.结果表明,可形成π-π共轭体系的芳香环,可显著增强磷酰化肽酯及小肽与氨基酸的相互作用力,π-π堆积力是首要的非共价相互作用力;可形成氢键的极性基团有利于形成复合物,但能否形成氢键还要受到分子柔顺性的影响;另外,分子大小、空间位阻对复合物的形成也有影响;而氨基酸的手性对它们的相互作用在质谱条件下没有表现出影响.     

聚乙烯醇与溶菌酶的相互作用及其对溶菌酶构象的影响

在生理条件下, 使用凝胶过滤色谱、荧光光谱法、差示扫描量热分析和傅里叶变换红外光谱法(FT-IR)研究了溶菌酶与聚乙烯醇(PVA)的相互作用. 结果表明PVA与溶菌酶结合形成复合物, 在它们的相互作用过程中, 溶菌酶酪氨酸的发射荧光部分被猝灭, 但是, 相互作用并没有改变酪氨酸的微环境; 差示扫描量热分析结果表明, 溶菌酶与PVA之间的相互作用没有破坏溶菌酶的高级结构; 进一步使用红外光谱法结合可增强分辨率的傅里叶去卷积技术和高斯曲线拟合技术共同用于对溶菌酶与PVA复合物冻干粉中溶菌酶酰胺I带的定量分析, 发现冻干粉溶菌酶分子中与分子间相互作用相关的β-折叠组分含量减少了, 但是, 用于衡量冻干状态蛋白质结构完整性的α-螺旋组分含量没有降低. 活性分析结果进一步确认, PVA与溶菌酶的相互作用没有破坏溶菌酶的三级结构.     

芳香胺与电子受体相互作用的吸收光谱研究

芳香胺与电子受体相互作用形成的电荷转移复合物(CTC)的吸收峰位置与两者的结构有关。弱的电子受体使芳胺紫外吸收峰加宽,而强的电子受体如四氰基乙烯(TCNE)与芳胺作用产生的CTC显示新峰。受体的受电子能力越强,新峰红移越多。芳胺苯环上氨基对位的取代基给电子能力越强,氮原子上烷基取代基越多,其与受体形成的CTC的吸收峰红移也越多。     

氮杂杯[2]-间/对-芳烃[2]三嗪与RDX氢键作用理论研究

利用MP2和mPWPW91方法,在6-311G**和6-311++G**基组水平上研究了RDX分别与硝基、氨基和迭氮基取代的氮杂杯[2]-间-芳烃[2]三嗪和氮杂杯[2]-对-芳烃[2]三嗪形成的分子间氢键相互作用,并借助自然键轨道(NBO)和分子中的原子(AIM)理论揭示了氢键的本质.结果表明,氮杂杯[2]-间-芳烃[2]三嗪复合物中氢键主要发生在RDX与三嗪环及其取代基之间;氮杂杯[2]-对-芳烃[2]三嗪复合物中氢键主要发生在RDX与杯芳烃环及其取代基之间.分子间相互作用能在-18.82~-40.62kJ/mol之间;经基组叠加误差(BSSE)校正后,相互作用能顺序为e>f≈b>a>c>d和e′>b′>f′>a′>d′>c′.两类复合物中,氨基取代的复合物分子间氢键强于硝基或叠氮基复合物分子间氢键,氨基氮杂杯[2]-对-芳烃[2]三嗪与RDX形成的氢键最强,有望作为降低火炸药感度、进行火炸药废水处理的候选物.为获得稳定性较强的RDX-氨基氮杂杯芳烃超分子炸药,应该选取介电常数较大的溶剂.     

二元和三元复合物中阳离子硫键与磷键的理论研究

采用MP2/cc-pVDZ和cc-pVTZ基组分别对复合物XH₂S⁺…NCH₂P和NCH₂P…PyX(X=NH₂, CH₃, H, CN, F, Cl, Br)中的硫键和磷键进行了研究, 讨论了键长、 键临界点的电荷密度(ρ)、 拉普拉斯密度(▽²ρ)、 范德华表面穿透距离、 二阶稳定化能和电荷转移量对硫键和磷键相互作用能的影响. 结果表明, 当取代基X为吸电子基团时, 形成的硫键较强. 当X为给电子基团时, 形成的磷键较强. 利用能量分解方法分析了取代基—CN导致硫键稳定性反常的可能原因. 还进一步讨论了三元复合物H₃S⁺…NCH₂P…PyX(X=NH₂, CH₃, H, CN, F, Cl, Br)中硫键和磷键的协同相互作用以及取代基对复合物稳定性的影响. 并通过对比相同的2种单体在三元复合物和二元复合物中的二阶稳定化能和相互作用能的差值, 说明了硫键与磷键起到相互促进的正协同作用, 增强了三元复合物的稳定性.     

Negative-ion electrospray ionization tandem mass spectrometry of N-phosphoryl amino acids and dipeptides

The negative-ions of N-phosphoryl amino acids were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The negative-ion ESI-MS/MS of N-phosphoryl amino acids showed characteristic fragmentation patterns different from those observed in the corresponding positive-ion ESI-MS/MS and negative-ion fast-atom bombardment mass spectra. For negative-ion ESI-MS/MS, a unique fragmentation from the N-terminal of N-phosphoryl amino acids or peptides containing a free beta-OH or CO(2)H group was observed to yield the characteristic fragment ion (RO)(2)P(O)O(-). The ease of the rearrangement depended on the position of the hydroxyl group in amino acids or peptides, and the N --> O rearrangement mechanism was proposed to involve the participation of the hydroxyl group. From previous solution-phase experiments and theoretical calculations, it was found that the beta-OH group was more active than gamma-OH, and the corresponding difference in negative-ion ESI-MS/MS was consistent with those previous findings.     

Quantitative determination of lysozyme-ligand binding in the solution and gas phases by electrospray ionisation mass spectrometry

Affinity constants for the binding of a range of substrate and non-substrate oligosaccharides to hen egg white lysozyme were determined by direct observation of the protein.ligand complexes using electrospray ionisation mass spectrometry (ESI-MS) with a chip-based nano-ESI source. The values obtained for a series of beta-1,4-N-acetylglucosamine oligomers (NAGn) were found to be in good agreement with those determined by fluorescence measurement. Oligomers of alpha-1,4-glucose (Glcn), which are believed to bind to lysozyme non-specifically, exhibited a 10(6)- to 10(8)-fold lower affinity for the enzyme. Lysozyme.NAGn complexes displayed an increase in Ka from n=2 to n=4, but then reached a plateau. In contrast non-specific lysozyme.Glcn complexes showed no such trend. Determination of gas-phase complex stability was achieved by quantitative collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) measurements. The collision energy (Ec50) or laser power (IRMPD50) required to dissociate precursor ions to 50% of their original intensity was determined for lysozyme.NAGn and Glcn complexes using the [M+8H]8+ charge state. An excellent correlation between trends in Ka and gas-phase stability was seen for NAGn oligomers bound to lysozyme, whereas no such relationship was observed with the non-specific, weaker lysozyme.Glcn complexes. These results illustrate that ESI-MS can be used to quantify the interactions between lysozyme and oligosaccharides in both the solution and gas phase and that measurement of gas-phase complex stability by CID or IRMPD can provide information about specific solution binding events.     

Study of complexes between lysozyme and sodium alkyl sulfates in the solid state

Complexes between lysozyme and sodium alkyl sulfates (decyl, dodecyl, tetradecyl and hexadecyl) in the solid state were prepared by mixing aqueous solutions of lysozyme and of the surfactant, separating the precipitated complex and purifying it. The stoichiometry of the complexes was investigated by elemental analysis and was found to correspond to about 8ǃ alkyl sulfate ions per lysozyme. Low- and wide-angle X-ray scattering were used to investigate the structure of the complexes. The scattering curves showed one fairly large scattering maximum, revealing a low state of organization of the complexes. The characteristic length characterizing the complexes was calculated from the value of the wave vector corresponding to the maximum of the scattered intensity. This length increased by about 0.233 nm per additional methylene group in the surfactant alkyl chain. A model where spherical aggregates of alkyl sulfate ions are arranged in a disordered simple cubic structure, dispersed in a matrix of lysozyme, provides a possible explanation of the results.     

The Investigation of Interaction Competition Between ATP and DIPP-Ala,Boc-Ala,or Ala by ESI-MS/MS and Theoretical Calculation

The interactions between ATP and N-(O,O-diisopropyl) phosphoryl-L-alanine (DIPP-Ala), N-(tert-butoxycarbonyl)-L-alanine (Boc-Ala), or L-alanine (Ala) were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The non-covalent complexes between ATP and Boc-Ala or DIPP-Ala were observed, while the complex between ATP and Ala was not found in the mass spectra. The affinity of DIPP-Ala for ATP was confirmed to be stronger than that of Boc-Ala by competition experiment. Through molecular modeling calculations, it was found that the non-covalent complexes were stabilized by intermolecular hydrogen bonds, and the affinity sequence for ATP was DIPP-Ala > Boc-Ala > Ala by comparing their binding energy, ?35.407 kcal/mol, ?15.634 kcal/mol, ?6.555 kcal/mol, respectively. The results implied that a phosphoryl group was a very important functional group to provide an interaction site between amino acids and ATP, and that N-phosphoryl amino acids can be used as a good model of protein in the studies of molecular recognition of ATP.     

Study on the interactions between ginsenosides and lysozyme under acidic condition by ESI-MS and molecular docking

In order to study the different effects of ginsenosides with similar structures, research on interactions between ginsenoside Rg1, Re and lysozyme was carried out by electrospray ionization mass spectrometry (ESI-MS) and molecular docking. The 1:1 and 2:1 noncovalent complexes of ginsenosides and lysozyme were observed in the mass spectra and the dissociation constants for them were directly calculated based on peak intensities of lysozyme and its noncovalent complexes with ginsenosides. The results showed that the 1:1 complex of ginsenoside Rg1 and lysozyme was more stable than that of ginsenoside Re and lysozyme. As the acidity increased, the stabilities of the 1:1 complexes of Rg1, Re and lysozyme both decreased. Interestingly, as the acidity increased, the stability of the 2:1 complex of Rg1 and lysozyme increased while that of Re decreased. From the result of molecular docking, ginsenosides interacted with the active sites of lysozyme. And the stability of the complexes could be affected by the conformation changes of lysozyme as acidity increased.     

Comb-like ionic complexes of cationic surfactants with bacterial poly(gamma-glutamic acid) of racemic composition

Comb-like ionic complexes, nATMA . PG(DL)GA, were prepared from microbial poly(gamma-glutamic acid), with a nearly racemic configuration, and alkyltrimethylammoniun bromides, with linear alkyl chains containing an even number of carbon atoms, n, ranging from 12 to 22. The complexes had a nearly stoichiometric composition, displayed thermal stability up to temperatures above 200 degrees C and were insoluble in water but soluble in organic solvents. In the solid state, they were arranged in a regularly layered structure with the alkyl side chain crystallized for n > or = 18. Heating above melting entailed a contraction in the interlayer distance which varied from 1 to 10% depending on the value of n. Comparison with data reported on similar complexes obtained from nearly enantiomerically pure poly(gamma-glutamic acid) revealed an overall behavior very similar for the two series but with specific significant differences concerning side chain crystallinity and dimensional response to temperature.     

Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.     

A novel vertical alignment technology for nematic liquid crystals based on electrostatic self-assembled method

A novel vertical alignment film for nematic liquid crystals is reported based on electrostatic self-assembly of alkyl sulfonic salts in aqueous solution. A series of self-assembled films with different alkyl chain lengths were prepared and used as alignment films. It was revealed that only when the number of carbon atoms in the alkyl chain approaches 11 or larger, could the self-assembled film induce vertical alignment of liquid crystals. We also found that the homeotropic alignment of liquid crystals was related to the surface roughness of self-assembled films. In addition, this vertical alignment film showed good electro-optical characteristics and excellent thermal stability.     

A novel vertical alignment technology for nematic liquid crystals based on electrostatic self‐assembled method

A novel vertical alignment film for nematic liquid crystals is reported based on electrostatic self‐assembly of alkyl sulfonic salts in aqueous solution. A series of self‐assembled films with different alkyl chain lengths were prepared and used as alignment films. It was revealed that only when the number of carbon atoms in the alkyl chain approaches 11 or larger, could the self‐assembled film induce vertical alignment of liquid crystals. We also found that the homeotropic alignment of liquid crystals was related to the surface roughness of self‐assembled films. In addition, this vertical alignment film showed good electro‐optical characteristics and excellent thermal stability.