巯基吡啶配基液相色谱固定相的制备及其对质粒的快速纯化

在基因治疗中,质粒是一种常用的非病毒型载体。由于目前市场上对质粒的需求量很大,因此有必要开发大规模的质粒制备技术。该文以双孔型流通色谱介质为基质,以巯基吡啶为配基,制得了液相色谱固定相,并考察了其对质粒DNA的纯化效果,分析了分离机理。结果表明:该分离介质对质粒的纯化是基于疏水作用;当上样量为10 mL (质粒质量浓度为0.30 mg/mL)、流速高达4 mL/min时,质粒仍然能以100%的收率实现纯化,其纯度为100%。该色谱介质对RNA的柱容量高达2.2 mg/mL。     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

双孔色谱介质的制备和质粒DNA快速色谱纯化

以甲基丙烯酸缩水甘油酯为单体,二甲基丙烯酸乙二酯为交联剂,环己醇与十二醇为有机致孔剂,200 g/L的碳酸钙悬浮液为内水相(超孔致孔剂),用二次乳化法制备了(W/O)/W乳液,通过紫外光引发悬浮聚合生成两类孔型高分子微球(DEA-B).DEA-B孔径为双峰分布,范围分别为50~200 nm和500~5000 nm.其体积平均粒径、湿密度与不含超孔的微孔介质(DEA-M)接近.在流速为0.5 mL/min(150 cm/h)及相同的洗脱条件下,比较了所制备的微孔色谱介质填充的色谱柱与双孔色谱介质填充的色谱柱对5.4 kb质粒DNA的分离效果.结果表明,仅双孔介质色谱柱可以纯化质粒DNA,并且可以在1500 cm/h的高流速下得到色谱纯的质粒DNA,证明该双孔色谱介质可以用于质粒DNA的高速分离.     

高效阴离子交换色谱-脉冲安培检测法测定烤烟中的水溶性葡萄糖、果糖和蔗糖

用高效阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）测定了烤烟中的水溶性葡萄糖、果糖和蔗糖。采用水浸取及膜过滤法处理烤烟样品,以Dionex CarboPac PA-1阴离子交换柱为色谱柱,0.2 mol/L NaOH水溶液为淋洗液进行分离测定。葡萄糖、果糖和蔗糖的含量与其峰面积的线性关系良好,回收率均在97%以上。方法简便易行,灵敏度高,重现性良好,可以实现对烟草中单糖的快速分离和测定。     

亲和色谱纯化超螺旋质粒DNA的研究进展

非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法。亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景。本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景。     

离子交换色谱在药用质粒分析中的应用

基因治疗和DNA疫苗在重大疾病的诊断和治疗方面具有广阔的前景,而分离过程的实时监测以及产品质量和规格的评定对于质粒制备过程开发、认证、产品批准至关重要。本文对阴离子交换色谱在药用质粒分析方面的应用与研究进展进行综述。目前应用于药用质粒制备过程流分析的离子交换色谱介质包括多孔、灌注、薄壳和非多孔等4种类型。文中概述了4类色谱介质在质粒和杂质以及质粒拓扑异构体分析中的应用现状并进行了对比,对于离子交换色谱分析方法在药用质粒分析过程中存在的问题以及发展趋势进行了评述。     

液相色谱法分离纯化烟草疫霉菌激发子

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离了一种新的９０ＫＤ激发子蛋白,他阴离子交换色谱流动相的最佳ｐＨ值,建立了疏水相互作用色谱,硫酸铵与三羟甲基氨基甲烷（Ｔｒｉｓ）缓冲液／水洗脱模式,简化了纯化步骤和活性损失的危险。     

阴离子交换色谱-积分脉冲安培检测法分离测定烟草料液中的山梨醇和糖

采用阴离子交换色谱-积分脉冲安培检测法分离测定了烟草料液中山梨醇、葡萄糖、果糖、蔗糖和麦芽糖,研究了山梨醇和糖在阴离子交换色谱中的保留行为。采用优化的水和氢氧化钠二元梯度淋洗条件,CarboPac PA10阴离子交换色谱柱进行分离,积分脉冲安培检测器检测一次进样测定烟草料液样品中的山梨醇、葡萄糖、果糖、蔗糖和麦芽糖。各组分在测试条件下线性关系良好,线性范围为0.005～20 mg/L,检测限为0.2～1.0 μg/L,加标回收率为95.1%~102.4%,相对标准偏差为1.2%～1.9%。     

强阴离子色谱法从毕赤酵母培养液中分离纯化重组巴西日圆线虫乙酰胆碱酯酶

提出了一种从基因工程毕赤酵母(Pichia pastoris)培养液中分离纯化重组巴西日圆线虫(Nippostrongylus brasiliensis)乙酰胆碱酯酶(NbAChE)的方法。采用Q-Sepharose Fast Flow强阴离子交换色谱柱对重组NbAChE进行了分离纯化。十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析表明,纯化物的活性峰为单一蛋白质带,其相对分子质量约为66000。该方法的活性回收率为52.6%,纯化因子为3.87;纯化后AChE的比活为 2837 U/mg。结果表明,该法是一种理想的分离纯化重组NbAChE的方法。     

离子色谱法测定浴盐中的阴、阳离子

用离子色谱法测定浴盐中的Na^ 、K^ 、Mg^2 、Ca^2 、Cl^-、Br^-、SO4^2-时，分离阳离子的色谱柱为ICS-C25阳离子交换柱，淋洗液为2.0mmol/L均苯四甲酸溶液，流速为0.6mL/min；分离阴离子时的色谱柱为shim-pack IC-Al阴离子交换柱，淋洗液为2.5mmol/L邻苯二甲酸溶液－2.4mmol/L三羟基氨基甲烷溶液（体积比为1：1），流速为1.0mL/min。所测离子Na^ 、K^ 、Mg^2 、Ca^2 、Cl^-、Br^-、SO4^2-在较宽浓度范围内有良好的线性关系，回收率为94.7%-102.4%，检出限为0.001-0.02mg/L，相对标准偏差为1.03%-1.63%。     

AOT/Triton X-100混合反胶束体系中假丝酵母脂肪酶催化蓖麻油水解的活性

研究了二(2-乙基己基)琥珀酸磺酸钠(AOT)/Triton X-100混合反胶束体系中假丝酵母脂肪酶(candida rugosa lipase)催化蓖麻油水解的反应. 考察了Triton X-100占总表面活性剂的摩尔分数(x(Triton X-100))、水与总体表面活性剂的摩尔比(ω0)、pH值、反应温度以及底物蓖麻油的浓度等因素对酶活性的影响. 研究结果表明, 加入非离子表面活性剂Triton X-100可以使假丝酵母脂肪酶的活性得到显著提高, 但是当底物蓖麻油的浓度大于0.24 mol·L-1时, 会对假丝酵母脂肪酶产生抑制作用.     

非离子表面活性剂层状液晶的结构与增溶作用

相行为研究表明，与ＴｒｉｔｏｎＸ－１００／Ｃ＿６Ｈ＿６／Ｈ＿２Ｏ体系相比，ＴｒｉｔｏｎＸ－１００／Ｃ＿（１０）Ｈ＿（２１）ＯＨ／Ｈ＿２Ｏ体系更易于生成层状液晶，并更为稳定，小角Ｘ－射线衍射表明；ＴｒｉｔｏｎＸ－１００／Ｃ＿（１０）Ｈ＿（２１）ＯＨ／Ｈ＿２Ｏ体系层状液晶中，Ｃ＿（１０）Ｈ＿（２１）ＯＨ与ＴｒｉｔｏｎＸ－１００交替排列于两亲双层中，Ｃ＿（１０）Ｈ＿（２１）ＯＨ／ＴｒｉｔｏｎＸ－１００重量比增加，两亲双层厚度ｄ＿０值不变。以Ｃ＿６Ｈ＿６代替Ｃ＿（１０）Ｈ＿（２１）ＯＨ后，Ｃ＿６Ｈ＿６存在于两亲双层的油层和渗入ＴｒｉｔｏｎＸ－１００分子链尾周围，使得溶剂渗透率减少，并且Ｃ＿６Ｈ＿６／ＴｒｉｔｏｎＸ－１００增加，ｄ＿０值增加。     

Triton X-100对血红蛋白/利巴韦林/H2O体系中血红蛋白性质和药物释放的影响

通过紫外光谱、荧光光谱、zeta电位、电导率、高效液相色谱和影像分析等方法研究了Triton X-100对血红蛋白/利巴韦林/H2O体系中血红蛋白性质和药物控制释放的影响. 研究结果表明, 随着Triton X-100浓度的增大, 荧光强度、荧光偏振、zeta电位和蛋白的形貌均发生较大的变化, Triton X-100能使增溶定位在蛋白表面中的利巴韦林逐渐游离出来. 当Triton X-100浓度大于1×10^-5 mol·L-1 时, Triton X-100 与血红蛋白的作用占主导地位, 血红蛋白开始明显变性. 少量Triton X-100能保护蛋白免受药物的影响.     

表面活性离子液体溴化N-十二烷基异喹啉与非离子表面活性剂Triton X-100在水溶液中的混合胶束

用等温滴定微量热法测定表面活性离子液体溴化N-十二烷基异喹啉([C₁₂iQuin]Br)与非离子表面活性剂Triton X-100混合物在水溶液中的临界胶束浓度。并以¹H核磁共振(NMR)和二维核Overhauser效应增强谱(2D NOESY)研究[C₁₂iQuin]Br与Triton X-100在混合胶束中的作用机理。研究结果显示：混合胶束中,Triton X-100分子的苯环定位于混合胶束的内核,聚氧乙烯链卷曲在异喹啉环周围。本文还应用规则溶液理论和浊点法对比研究了十二烷基三甲基溴化铵(DTAB)-Triton X-100混合胶束体系的相关性质。     

Effects of Triton X-100 on Properties of Hemoglobin and Controlled Release of Drug in Hemoglobin/Ribavirin/H2O System

Effects of Triton X-100 on the properties of hemoglobin (Hb) and on the controlled release of ribavirin were studied using the methods of UV-Vis spectrum, fluorescence spectrum, zeta potential, conductivity, high-performance liquid chromatographic (HPLC), and image morphology in Hb/ribavirin/H₂O system. With the increase of concentration of Triton X-100 in the system, the intrinsic fluorescence intensity, synchronous fluorescence intensity, fluorescence polarization, zeta potential, and morphology of Hb all changed gradually, and the ribavirin located on the Hb surface was dissociated and released out. When the concentration of Triton X-100 was higher than 1×10⁻⁵ mol·L⁻¹, the stronger interaction of Triton X-100 with Hb was predominant. Hb was unfolded and denaturized. A little Triton X-100 can protect Hb from the effects of ribavirin.     

Clouding Behavior and Thermodynamic Study of Nonionic Surfactants in Presence of Additives

The present study investigates the effect of different additives on the cloud point (CP) of nonionic surfactants Triton X-100 (TX-100) and Triton X-114 (TX-114) in aqueous solutions. The thermodynamic parameters of these mixtures were calculated at different additive concentrations. The cloud point of nonionic surfactants TX-100 and TX-114 decreased with the increment of electrolyte concentrations and increased with alcohol concentrations. The standard Gibbs free energy was found to be positive for both the surfactants, whereas the enthalpy and the entropy of the clouding phenomenon were found to be positive with alcohols and negative with electrolytes. The overall clouding process was endothermic for alcohols and exothermic for electrolytes.     

Inhibition of Triton X-100／n-C5H11OH／H2O System on Hydrolysis of Cephanone

The hydrolysis of cephanone in Triton X-100 micelle and Triton X-100/n-C5H11OH/H2O(O/W) microemulsion were studied by means of UV-Vis absorption spectroscopy. The results show that compared with water, Triton X-100 micelle and Triton X-100/n-C5H11OH/H2O (O/W) microemulsion can inhibit the hydrolysis of cephanone. The inhibition effects of Triton X-100 micelle and Triton X-100/n-C5H11OH/H2O (O/W) microemulsion on the hydrolysis of cephanone are related to the location of cephanone in the interphases of Triton X-100 miceUes and Triton X-100/n-C5H11OH/H2O(O/W) microemulsion droplets.     

Elution of 17α 25-Norhopanes and Triaromatic Steroids from Weathered Soils by Mixed Triton X-100/Na2SiO3 Surfactant Solution

Sodium silicate(Na₂SiO₃) was used to improve the elution of super heavy oil from weathered soil on an ultrasound-enhanced elution system by the solution containing 0―6000 mg/L surfactant Triton X-100. The removal extent of three markers[C_26―34 17α 25-norhopanes, C_26―28 triaromatic steroids(TAS), and C_27―29 methyl triaromatic steroids(MTAS)] was monitored. The average elution percentages of C_26―34 norhopanes, C_26―28 TAS, and C_27―29 MTAS by Triton X-100/Na₂SiO₃ solutions were increased by 11%―13%, 9%―11% and 8%―13% with increasing Triton X-100 concentrations from 150 mg/L to 6000 mg/L. All the concentrations of Triton X-100 improved the elution of TAS homologs containing fewer carbon atoms, whereas high concentrations improved the elution of larger 17α 25-norhopane and MTAS species. Addition of Na₂SiO₃ produced a noticeable increase in elution, particularly for lower-weight species. Scanning electron microscope(SEM) images and energy spectroscopy data reveal that surfactant solution of 6000 mg/L Triton X-100 and 4000 mg/L Na₂SiO₃ produced the greatest improvement in the elution of super heavy oil aggregates encapsulating the soil surface and the emulsification of particle dispersions. That is to say mixed solutions of Triton X-100 and Na₂SiO₃ in combination with ultrasound are a potential means of removing super heavy oil from weathered soils.     

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.     

Cloud point extraction equilibrium of lanthanum(III), europium(III) and lutetium(III) using di(2-ethylhexyl)phosphoric acid and Triton X-100

The cloud point extraction behaviors of lanthanoids(III) (Ln(III) = La(III), Eu(III) and Lu(III)) with and without di(2-ethylhexyl)phosphoric acid (HDEHP) using Triton X-100 were investigated. It was suggested that the extraction of Ln(III) into the surfactant-rich phase without added chelating agent was caused by the impurities contained in Triton X-100. The extraction percentage more than 91% for all Ln(III) metals was obtained using 3.0 × 10⁻⁵ mol dm⁻³ HDEHP and 2.0% (v/v) Triton X-100. From the equilibrium analysis, it was clarified that Ln(III) was extracted as Ln(DEHP)₃ into the surfactant-rich phase. The extraction constant of Ln(III) with HDEHP and 2.0% (v/v) Triton X-100 were also obtained.