Synthesis of Novel Phosphorylated Daidzein Derivatives and ESI Investigation on Their Non-Covalent Complexes with Lysozyme

Daidzein （7,4＇-dihydroxyisoflavone） was phosphorylated by a modified Atherton-Todd reaction. The structures of the five target product, were determined by X-ray, IR, NMR and ESI-MS. Electrospray ionization results show that in the gas phase all the phosphorylated daidzein derivatives could form non-covalent complexes with the protein lysozyme, while non-covalent complexes were not detected in the mixed solution of daidzein with lysozyme. Relative affinity of every non-covalent complex was obtained according to its different decomposition orifice voltage.     

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.     

蛋白质与四氰代二甲基苯醌荷移反应的研究

用分光光度法研究了蛋白质与四氰代二甲基苯酪(TCNQ)之间的荷移反应．通过 对影响反应因素的研究，确立了以形成荷移(CT)配合物测定蛋白质的最佳反应条件 ，在V(丙酮)：V(水)＝1：4，pH＝9．8的溶液中，配合物在425nm处有最大吸收， BSA在0—40ug/mL及50—300ug/mL符合朗伯—比尔定律，摩尔吸光系数分别为2． 43×10^5L．mol^-1.cm^-1和2．90×10^5L.mol^-1.cm^-1。分别用平衡透析法、双 波长法和摩尔比法研究了蛋白质与TCNQ的结合方式并测定了最大结合数．当TCNQ浓 度较小时，与蛋白质的结合符合Scatchard规则，存在两类结合方式，具有不同结 合常数；TCNQ浓度较高时，符合Plasvento相分配模型，分配常数为一定值．用拟 合的方法测定了人血清样品中的蛋白质，与考马斯亮蓝法测定结果吻合，回收率为 98％—104。     

一体化平台石墨炉原子吸收光谱法直接测定全血中顺铂

用ψ（HNO3）=0．5％的硝酸稀释样品，一体化平台石墨炉原子吸收光谱法直接测定全血中顺铂，无须进行基体匹配，Pt的特征质量m=59pg，检出限为32pg(3σ)，相对标准偏差(RSD，n=7)小于5％，样品加标回收率在95％～105％之间。     

Active bead-linked immunoassay on protein microarrays

Protein microarrays are becoming a powerful tool in proteome, biochemical, and clinical studies. In addition to the quality of arrayed immobilized probe molecules, sensitivity of the microarray-based assay is highly dependent on the detection technique. Here we suggest four simple techniques for rapid detection of analytes bound to protein microarrays. The techniques employ functionalized magnetic and non-magnetic beads moved to, from, or along the array surface by external forces. In contrast to other labeling techniques actively controlled physical labels: (i) make detection extremely fast to allow microarray reading in seconds; (ii) provide a low background due to active removal of weakly bound beads; and (iii) provide a highly sensitive detection, since one antigen-antibody bond is capable of holding bead immobilized on the array surface. In combination with the electrophoretically assisted active immunoassay we described recently such active reading allows to reduce total indirect immunoassay time to 7-10 min while having sensitivity in the femtomolar concentration range. High speed, sensitivity, and specificity make active bead-linked detection an ideal choice in rapid high-throughput screening and in emergency diagnostics.