毛细管电泳技术分析水稻中的总核糖核酸

 通过高温干烤和焦碳酸二乙酯 (DEPC)两种技术除去核糖核酸 (RNA)酶 ,在保证RNA的稳定性的情况下 ,在1.0 %T ,0 %C的线性聚丙烯酰胺 (即丙烯酰胺和双丙烯酰胺的总质量分数是 1.0 % ,交联度为 0 % )筛分介质中 ,利用无胶筛分毛细管电泳技术对水稻中的总RNA进行了分析 ,整个分析过程在 15min内完成。利用 5 .0 %T ,0 %C线性聚丙烯酰胺筛分介质 ,对水稻中的转移RNA(tRNA)进行了较为详细的分析 ,在 30min内可将tRNA分为两组共 9个峰。该方法可用于植物中RNA的分析 ,具有快速、准确的特点。     

偶氮胂Ⅲ—Ba(Ⅱ)共振散射光谱法测定人血清蛋白

在pH 3.0~3.8的BR缓冲溶液中,偶氮胂Ⅲ-Ba(Ⅱ)与蛋白质结合形成复合物,导致体系的共振散射信号增强,据此建立了蛋白质测定的共振散射光谱法。结果表明,在优化的实验条件下,牛血清白蛋白(BSA)的线性范围为0~1.0 mg/L,检出限为21.4μg/L;将该法用于人血清中蛋白质的测定,与医院测定结果基本一致;方法的批内精密度为2.8%(n=6),批间精密度为3.5%(n=6),回收率为96.0%~100.0%。     

Mo(Ⅵ)-邻硝基苯基荧光酮配合物光谱探针测定蛋白质

研究了Mo(Ⅵ)-邻硝基苯基荧光酮(o-NPF)配合物与蛋白质的相互作用及光谱性质. 提出了以Mo(Ⅵ)-邻硝基苯基荧光酮配合物作为光谱探针测定微量蛋白质的方法. 结果表明, 在pH为2.2的 HAc-NaAc缓冲介质中, 乳化剂OP存在下, o-NPF-Mo(Ⅵ)在40 ℃加热10 min后与蛋白质形成稳定的复合物, 最大吸收波长红移至587 nm. 蛋白质质量浓度在0～10 mg/L 范围内符合比尔定律, 复合物的表观摩尔吸光系数为3.53×106 L·mol-1·cm-1. 该方法具有很高的灵敏度和选择性, 可直接用于人尿、新生牛血清中蛋白质的测定, 回收率分别为93%和99%.     

蛋白质与罗丹明B相互作用的极谱分析

在pH 5.0的六次甲基四胺缓冲液中,罗丹明B能够与蛋白质相互作用形成复合物。该复合物使罗丹明B在-0.87 V处的极谱还原峰峰电流下降,在实验选定的最佳条件下,该峰峰电流的下降值同牛血清白蛋白(BSA)的含量在2.5~30 mg/L范围内呈线性关系,检出限为1.3 mg/L。该法已应用于血清样品中蛋白质的测定,分析结果满意。     

基于可断裂三功能探针的酪氨酸磷酸化蛋白质复合物的规模化分析

在细胞的信号转导过程中,磷酸化酪氨酸（Phosphotyrosine, pTyr）信号在许多通路中起着重要的调控作用。受pTyr信号调控的蛋白质复合物是信号传递早期过程中的关键分子机器,分析该类动态蛋白质复合物具有重要的意义。Photo-pTyr-scaffold是一种结合了化学探针与pTyr识别蛋白质结构域的化学蛋白质组学分析策略,采用该策略已经实现了对表皮生长因子受体（Epidermal growth factor receptor, EGFR）通路相关的pTyr信号蛋白质复合物的规模化分析。但是,该方法无法解析所鉴定复合物的一一对应关系,增加了后续复合物解析的复杂性。本研究在Photo-pTyr-scaffold探针设计的基础上,将可断裂基团引入至其生物素富集端,设计合成了新型的可断裂三功能探针,以实现对富集产物的可控洗脱。基于pTyr信号蛋白质复合物在化学交联后的分子量差异,利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳实现了对洗脱的pTyr蛋白质复合物的分离,进一步将不同分子量区域的蛋白质进行胶内酶解和质谱分析。理论上,发生化学交联的pTyr蛋白质复合物将在高于原蛋白质分子量的区域被...     

纳米Au/核酸适配体/硅量子点荧光传感体系用于黄曲霉毒素B1分析检测

以自组装方法,构建了Au纳米粒子(AuNPs)/寡核苷酸/硅量子点(SiQDs)复合物荧光传感体系,AuNPs使复合物中咪唑基硅量子点荧光猝灭。样品溶液中存在黄曲霉毒素B1(AF B1)时,复合物中寡核苷酸与AF B1发生特异性反应,释放出咪唑基硅量子点,使体系的荧光得到恢复,并且其荧光强度随加入样品中AF B1量的增大而增强。优化实验条件为缓冲溶液pH=7.5,50 mmol/L NaCl,孵化时间5 min。在最优条件下,AF B1浓度在0.01～1.0 ng/mL范围与体系荧光强度恢复程度呈现出良好的线性关系,方法检测限(3σ)为8 pg/mL。该方法已成功应用于实际样品中AF B1的测定,其回收率在94.0%～106.0%范围,相对标准偏差为2.1%～3.5%。     

高效液相色谱法测定方便面中丙烯酰胺

建立了一种检测方便面中丙烯酰胺的液相色谱方法。萃取剂选择1g/L的蚁酸溶液,将萃取液以12000r/min,2℃离心18min,上清液再以14500r/min,0℃离心18min,可分离基质中淀粉、蛋白质、脂肪等干扰物;方法采用ZORBAX SB-C18(250mm×4.6mm,5μm)色谱柱,流动相为A(乙腈∶水=1∶24(V/V))和B(乙腈),流速0.5mL/min,在丙烯酰胺的最大吸收197nm处检测,提高了方法的检出限及回收率。该方法有良好的线性关系(r=1.00000),检出限为48.0μg/kg。回收率94.4%～109.8%,相对标准偏差为4.9%～5.6%。     

极端嗜盐古生菌启动子序列缺失突变的微量热研究

用微量热方法和DNA缺失突变技术研究了来源于极端嗜盐古生菌R1上的一个推测的启动子片段(RM10)在大肠杆菌中的启动子功能. 启动子片段融合到质粒pKK232-8上无启动子的氯霉素乙酰转移酶(CAT)基因前来检测它驱动基因表达的能力, 缺失分析RM10启动子片段定位具有启动活性的重要功能区. 实验结果从热动力学角度揭示, 这个启动子片段上含有－35区和－10区特征的1382～1517 bp(碱基对)区段是它在大肠杆菌中具有启动子功能的关键部分; 在1～1382 bp区段或1571～1848 bp区段上还存在它的负调控区. 该研究为基因启动子功能研究提供了一种新的、更加灵敏便捷的、化学与生物学相结合的方法.     

基于葡萄糖和苯硼酸基元之间的可逆共价键构筑多重响应性高分子复合物胶束

通过可逆加成-断裂链转移(RAFT)的聚合方法,合成了分别含有苯硼酸基元和葡萄糖基元的聚(N-异丙基丙烯酰胺)-b-聚(丙烯酰胺基苯硼酸)(PNIPAM-b-PAPBA)和聚(N-异丙基丙烯酰胺)-b-聚(丙烯酰葡萄糖胺)(PNIPAM-b-PAGA)二嵌段聚合物.由于苯硼酸和葡萄糖基元之间在弱碱性条件下(pH9.3)形成硼酸酯共价键,两种二嵌段聚合物的水溶液混合后能自发形成以PAPBA/PAGA络合物为核,PNIPAM为壳层的高分子复合物胶束.由于硼酸酯共价键在pH值和葡萄糖浓度改变时能可逆形成和断裂,以及胶束PNIPAM壳层的温敏性,所制备的基于苯硼酸/葡萄糖可逆共价键的高分子复合物胶束对pH、葡萄糖和温度具有多重响应性.     

可生物降解肝素钠/两性壳聚糖复合物用于蛋白药物pH响应释放研究

制备了一类可生物降解肝素钠两性壳聚糖复合物(HPACS),并探索将其用于蛋白药物pH响应释放.两性壳聚糖由壳聚糖与丙烯酸加成反应得到,丙烯酸取代度可通过丙烯酸壳聚糖投料比调控;用胶体与pH浊度滴定研究了肝素钠与两性壳聚糖的复合作用,发现两组分在一定pH范围内能通过静电相互作用形成复合物,复合转变临界pH(pHΦ)与两性壳聚糖中丙烯酸取代度有关,取代度越低,pHΦ值越高.以牛血清白蛋白(BSA)为模型,测定了其在复合物中包埋及不同pH介质中的释药行为.结果表明,BSA可以在非常温和条件下有效包埋于复合物中,包埋率接近100%;BSA从复合物中释放具有很高的pH响应性,释放转变在很窄的pH范围内(<0.4pH单位)完成,释放转变临界pH(pH′Φ)可由两性壳聚糖中丙烯酸取代度调控.复合物形成和蛋白质释放在对pH依赖性上存在很好的相关性.同时还发现,在中性介质中(pH7.4),复合物对BSA具有很好的缓释作用,BSA持续释放时间可达15天左右.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique

In this work, two high‐performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and L ‐glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o‐phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C₁₈ column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate–acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)–methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

毛细管区带电泳分析五步蛇毒蛋白C激活剂和蛋白C的相互作用

建立了毛细管区带电泳分析五步蛇毒蛋白C激活剂(protein C activator, PCA)与血浆蛋白C(protein C, PC)的相互作用。以未涂层毛细管(60.2 cm(有效长度50 cm)×75 μm)为分离柱,50 mmol/L Tris-HCl(pH 7.4)为运行缓冲液,于198 nm波长下检测。对影响五步蛇毒PCA分离的因素(如缓冲液、离子浓度)及在37.5 ℃下孵育不同时间的五步蛇毒PCA与PC的相互作用进行考察。方法的检出限(以信噪比为3计)为3 mg/L,线性范围为10～300 mg/L。五步蛇毒PCA迁移时间和峰面积的相对标准偏差分别为0.56%和3.8%(n=6)。等体积的五步蛇毒PCA(200 mg/L)与PC(60 mg/L)孵育5 min,其结合率达到最大,且谱图中未见有PC水解的肽链。该五步蛇毒PCA可改变PC空间构象直接激活PC。该方法简单,灵敏度和分辨率高,分析结果为今后快速检测五步蛇毒PCA及其活性提供了重要的理论依据。     

应用随机寡核苷酸文库修饰磁性颗粒提高血浆蛋白质的鉴定覆盖度

基于随机寡核苷酸与蛋白质之间的离子、亲和、疏水、氢键等相互作用力及多种空间结构作用,发展了一种基于随机寡核苷酸文库作为配基的新型血浆样品处理方法。采用寡核苷酸文库修饰的磁性颗粒(MNP@ssDNA)材料,在生理缓冲体系条件下捕获血浆样品中的蛋白质。比较了两种洗脱体系的洗脱效果,并利用nano-RPLC-ESIMS/MS对获得的蛋白质酶解液组分进行分析。结果表明,MNP@ssDNA材料处理后的血浆蛋白质鉴定数量提升了约29.5%,两种洗脱体系呈现良好的互补性(26.7%)。血浆中前10种高丰度蛋白质的谱图占有率从处理前的31.82%降低到21.31%(洗脱体系1)和26.20%(洗脱体系2)。在鉴定到的蛋白质中,丰度最低的蛋白质在血浆中的质量浓度约为0.29 ng/mL,该蛋白质仅在MNP@ssDNA材料处理后被鉴定到。结果证明MNP@ssDNA策略不仅能有效降低血浆中高丰度蛋白质的丰度,也为低丰度蛋白质的深度挖掘提供了新的思路。     

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the α subunit of Gs (GαsQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GαsQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GαsQL-stimulated Bak reporter luciferase activity. Expression of GαsQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Gαs activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Gαs augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.     

Thermodynamic Study of Myelin Basic Protein upon Interaction with [Hg2+] Using Extension Solvation Model

Mercury ion interaction with myelin basic protein (MBP) was studied at 300 K in 30 mmol/L tris buffer, pH=7 by isothermal titration calorimetry (ITC). An extended solvation model was used for Hg²⁺+MBP interaction over the whole range of Hg²⁺ concentrations. The binding parameters recovered from the solvation model were attributed to the structural changes of MBP due to its interaction with mercury ion. It was found that mercury ion acted as a noncooperative effector of MBP, and there is a set of two identical and independent binding sites for Hg²⁺ ions. The dissociation equilibrium constant is 97.6 µmol/L. The molar enthalpy change of binding is ?11.25 kJ·mol^?1.     

Sensitive profiling of biogenic amines in human urine by capillary electrophoresis with field amplified sample injection

In order to monitor biogenic amines in human urine, a method based on field‐amplified sample injection combined with capillary electrophoresis and direct UV absorption detection was developed. Dopamine, tyramine, tryptamine, serotonin and epinephrine were effectively separated and identified in human urine samples, and detection limits were 0.072, 0.010, 0.027, 0.010 and 0.120 µmol/L, respectively. Detection limits comparable to laser‐induced fluorescence detection or solid phase extraction combined with capillary electrophoresis were achieved. Parameters affecting electrophoretic system detection sensitivity were investigated. Optimal separation conditions were obtained using as background electrolyte a pH 6.5 mixture of 2‐(morpholino)ethanesulfonic acid 20 mmol/L and 30 mmol/L phosphate buffer, containing 0.05% hydroxypropylcellulose and 10% v/v methanol. Injections of the sample solution were performed by applying a voltage of 12 kV for 50 s. Recovery and accuracy ranged between 89.4 and 94.9%, and 89 and 112%, respectively. The method was successfully applied on actual urine samples (from a healthy volunteer): target bioamine content was consistent with endogenous levels reported in the literature. The proposed method is simple, fast and inexpensive and can be conveniently employed in work‐related stress studies. The affordability and noninvasive sampling of the method allow epidemiological studies on large number of exposed persons to be performed. Copyright © 2013 John Wiley & Sons, Ltd.     

三氟拉嗪与八肋游仆虫中心蛋白N端半分子的结合及对蛋白功能的影响

采用荧光光谱、 圆二色光谱(CD)、 等温滴定量热分析(ITC)、 电泳及分子对接等分析技术, 研究了三氟拉嗪(TFP)与八肋游仆虫中心蛋白N端半分子(apoN-EoCen)的结合, 考察了TFP对apoN-EoCen性质的影响. 结果表明, 在室温下10 mmol/L Hepes缓冲溶液(pH=7.4)中, TFP与apoN-EoCen以摩尔比1∶1结合于apoN-EoCen的第二个EF-手的E, F螺旋之间, 条件结合常数约为10 ³ L/mol; TFP的结合导致蛋白质二级结构发生改变, α螺旋含量减小, Tb ³⁺敏化荧光强度降低83%, apoN-EoCen切割DNA的类核酸酶活性明显受到抑制; Tb ³⁺仍可占据复合物apoN-EoCen-TFP中蛋白质的2个金属离子结合位置, 条件结合常数约为7.0×10 ⁵ L/mol, TFP的结合不抑制金属离子诱导的蛋白质聚集.