大孔树脂吸附与反相色谱纯化恩拉霉素

恩拉霉素作为多肽类抗生素,是一种新型、安全的饲料添加剂。本文建立了一条基于大孔树脂初纯和反相色谱精制的分离纯化工艺。该工艺路线首先使用AB-8大孔树脂在0.012 mol/L盐酸溶液-甲醇(50:50,V/V)缓冲液条件下洗脱实现恩拉霉素初步纯化,再使用制备型C18反相色谱柱在0.05 mol/L磷酸二氢钠-乙腈(70:30,V/V)(p H 4.5)缓冲液洗脱下实现恩拉霉素a和b的有效分离,a、b两个组分纯度分别达到98.5%和98.0%,a和b两种有效成分的总收率为29.2%。本研究为恩拉霉素a和b两种纯品的制备以及高纯度恩拉霉素产品的生产提供了参考。     

总神经节苷脂的简便纯化方法

许多研究者先后提出了多种分离纯化神经节苷脂(Gls)的方法,其中Yu等建立的相继用离子交换剂、吸附剂分别层析的方法在国内较为常用,但其周期较长、步骤较繁。本文将离子交换剂与吸附剂装入同一柱内一次完成Gls的层析纯化,并用含盐氯仿(C)-甲醇(M)混合液(C/M=2/7,V/V,含0.1mol/L NaAc)代替Yu法B液进行洗脱,用国产硅胶代替价格昂贵的Iatrobeads,取得了良好的效果。本法的优点是能节省时间及有机溶媒。     

Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究

Rhodococcus ruber CGMCC309菌株为酰胺酶及腈水解酶双重缺陷菌株,研究表明该菌能产宽泛底物特异性的腈水合酶。对该菌株产生的新型腈水合酶(NHase-3090)进行纯化和结晶,并研究了其酶学性质。采用疏水、离子交换及凝胶过滤3种层析方法,使该酶纯化倍数达到17.14,得率高达26.2%。电泳分析表明,全酶分子量为105 kDa,由α(24.3 k Da)和β(28.0k Da)2个亚基组成,并构成α2β2四聚体。酶的最适p H和温度分别为7.5和30℃。该酶明显受不同金属离子影响。动力学研究表明,Km为178.8 m M;Vmax为209.1μmol/L·min·mg。研究发现3种金属离子Zn~(2+),CO~(2+)和Cd~(2+)有利于酶蛋白结晶。结晶最佳条件是:采用112-34#试剂(0.05mol/L水合硫酸镉、0.1mol/L HEPS和1.0mol/L三水醋酸钠),蛋白质浓度为15 mg/ml,结晶温度为16℃,p H为7.5,结晶时间为30 d。腈水合酶蛋白单晶经X射线衍射,分辨率达到了3.7。该腈水合酶的纯化和结晶为进一步深入研究其结构和功能奠定了基础。     

从蚯蚓中联合提取抗氧化酶SOD、CAT的方法研究

本研究采用闪式提取技术,固液比为1:4(m/V)的2.5 mmol/L pH 7.0磷酸缓冲液,提取转速5500 rpm,提取时间2 min,从蚯蚓体内提取出SOD、CAT,并通过羧甲基纤维素CM-22离子交换层析实现SOD和CAT的联合提取分离,SOD、CAT的活性回收率分别达到88.23%和69.5%。在纯化工艺中经过丙酮沉淀和柱层析技术得到蚯蚓SOD纯品,比活达到9352 U/mg,产物在SDS-PAGE上为单一条带,其亚基分子量约为17 kD;通过柱层析纯化了蚯蚓CAT,比活达到22606 U/mg。     

跨膜蛋白AgrC_(TM6-7C)分离纯化方法的优化北大核心CSCD

旨在通过优化AgrC_(TM6-7C)蛋白的纯化过程,获得高纯度的目的蛋白,为后续研究提供材料。详细探讨了金属离子螯合层析缓冲液中咪唑浓度、离子交换层析缓冲液的pH值对AgrC_(TM6-7C)蛋白纯化效果的影响,并研究了β-巯基乙醇对AgrC_(TM6-7C)蛋白聚集及其激酶活性的影响。经过优化后,在不影响AgrC_(TM6-7C)蛋白激酶活性的前提下,AgrC_(TM6-7C)蛋白在浓度和纯度上均有明显提高,纯度可达98%以上,产量可达15 mg/L。     

层析法纯化破伤风毒素的试验研究

为了获得高纯度的破伤风毒素,用疏水层析和离子交换层析纯化破伤风毒素。破伤风毒素培养滤液经Phenyl Sepharose疏水层析除去大部分杂质,再经DEAE Sephadex离子交换层析进一步纯化。经两步层析纯化后,毒素纯度达到2000Lf/mg PN以上,回收率为52%~73%。用此方法,连续纯化五批毒素,均获得高纯度的破伤风毒素。试验证明破伤风毒素经疏水层析和离子交换层析可得到有效纯化。     

白眉蝮蛇蛇毒神经生长因子的研究：Ⅰ.神经生长因子（NGF）的纯化及其生物学？

研究白眉蝮蛇蛇毒神经生长因子纯化方法并行生物学活性鉴定。方法 从白眉蝮蛇乌苏里亚种蛇毒中,经硫酸胺分段盐析,提取含有神经生长因子的粗制品,再经二乙氨基乙基纤维素离子交换层析和羧甲基纤维素离子交换层析,分离出蛋白质纯品,经十二烷基磺酸钠－聚丙烯酰胺凝胶电泳进行鉴定。     

跨膜蛋白AgrC_(TM6-7C)分离纯化方法的优化

旨在通过优化AgrC_(TM6-7C)蛋白的纯化过程,获得高纯度的目的蛋白,为后续研究提供材料。详细探讨了金属离子螯合层析缓冲液中咪唑浓度、离子交换层析缓冲液的pH值对AgrC_(TM6-7C)蛋白纯化效果的影响,并研究了β-巯基乙醇对AgrC_(TM6-7C)蛋白聚集及其激酶活性的影响。经过优化后,在不影响AgrC_(TM6-7C)蛋白激酶活性的前提下,AgrC_(TM6-7C)蛋白在浓度和纯度上均有明显提高,纯度可达98%以上,产量可达15 mg/L。     

海洋扩展青霉（Penicillium expansum）果胶酶的纯化及酶学性质研究

通过超滤、DEAE Sephadex A-50离子交换层析、Sephadex G-100凝胶过滤层析,对一株来自海洋的扩展青霉(Penicillium expansum)所产果胶酶进行分离纯化,得到电泳纯的果胶酶,经SDS-PAGE电泳显示单一条带,且果胶酶亚基的分子质量约为63.96 ku,纯化倍数为24.13,回收率为36.32%。酶学性质研究结果表明,该果胶酶的最适反应温度为50℃,最适p H值5.4,在p H值4.6~6.2比较稳定,Mg2+、Ca2+对果胶酶活力有明显激活作用,Cu2+有明显抑制作用,以果胶粉为底物的Vmax为393.56μg/(min·m L),Km为3.34 mg/m L。     

白眉蝮蛇蛇毒神经生长因子的研究Ⅰ.神经生长因子(NGF)的纯化及其生物学活性鉴定

研究白眉蝮蛇蛇毒神经生长因子纯化方法并行生物学活性鉴定。方法从白眉蝮蛇乌苏里亚种蛇毒中,经硫酸胺分段盐析,提取含有神经生长因子的粗制品,再经二乙氨基乙基纤维素离子交换层析和羧甲基纤维素离子交换层析,分离出蛋白质纯品,经十二烷基磺酸钠——聚丙烯酰胺凝胶电泳进行鉴定,结果纯化的纯品为单一区带,分子量为４３ｋｕ。生物学活性鉴定证明分离出的该蛋白质纯品可促进神经细胞突起密集生长。     

固相萃取-高效液相色谱法测定复合薯片中丙烯酰胺

建立了一种利用固相萃取小柱与高效液相色谱联用,采用光电二极管阵列检测器测定复合薯片中丙烯酰胺的方法。以0．1％的甲酸水溶液为提取试剂,采用Waters Oasis HLB固相萃取小柱（200mg／6cc）对提取液净化处理,然后以流动相为甲醇／水（5：95,V：V）,流速为0．6mL／min,检测波长为210nm,利用AgilentC18反相色谱柱进样分析。在该条件下丙烯酰胺的回收率达80％以上,最低检测限小于10ng／mL。该方法操作简便、重复性好、稳定性高,可用于油炸复合薯片中丙烯酰胺的快速检测。     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

甲氨基阿维菌素苯甲酸盐微乳剂热贮稳定性研究

采用高效液相色谱法分析甲维盐微乳剂,以甲醇＋0．2％三乙胺溶液＋乙腈（V：V：V=30：30：40）为流动相,采用XterraRP184．6150mm色谱柱,检测波长245nm,测得54±2℃条件下14d分解率为3．42％。室内毒力测定热贮前后1％甲维盐微乳剂Lc50分别为0．499mg／L和0．524mg／L,热贮分解率为4．78％,与高效液相色谱测定结果基本一致。     

高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。     

大孔吸附树脂结合硅胶柱层析纯化环孢菌素A

采用大孔吸附树脂层析结合硅胶柱层析,对环孢菌素A的分离纯化进行研究,确定了最佳层析条件,建立了工业化制备环孢菌素A的工艺。大孔吸附树脂层析选用D101树脂作为吸附介质,提取液丙酮含量控制在50%,最大吸附量为35 mg/g湿树脂,洗脱剂选用丙酮;硅胶柱层析选用42~64μm硅胶作为层析介质,最优层析条件为柱床高径比10∶1,流动相配比V(石油醚)∶V(丙酮)=70∶30,流速80 mL/m in,环孢菌素A上样质量浓度100 g/L,硅胶层析平均收率为84.2%,环孢菌素A纯度可达到97%以上,整个工艺总收率为65%~70%。     

用疏水色谱法复性并同时纯化重组人粒细胞-巨噬细胞集落刺激因子

目的：建立一种简单、快速复性并同时纯化大肠杆菌表达的重组人粒细胞一巨噬细胞集落刺激因子（rhGM-CSF）的方法。方法：研究rhGM-CSF在疏水色谱（HIC）上的复性和纯化机理,并对固定相和流动相进行选择和优化,包括固定相配基、流动相中盐的种类、流动相pH值、流动相中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的比例,以及流动相中尿素的浓度。结果：优化后的固定相为PEG600,流动相中的盐为（NH4）2SO4,流动相pH值为7．0,流动相中添加2．0mol／L尿素、1．8mmol／LGSH和0-3mmol／LGSSG。在优化条件下,HIC可使rhGM-CSF在分离纯化的同时得到复性,比活达1．58×10^7U／mg,纯度为95．7％,质量回收率为56．8％。结论：建立的疏水色谱复性和纯化工艺可简化操作步骤,缩短生产周期。     

Two-dimensional high-performance liquid chromatographic determination of 5-hydroxyindoleacetic acid and homovanillic acid in urine

The biomedically and neurochemically important compounds 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) have been simultaneously determined in human urine after reverse-phase two-dimensional high-performance liquid chromatography. A 10-fold-diluted urine sample (20 microliters) is first separated on a C18 column (30 X 0.39 cm) using an 85% pH 6.0 phosphate buffer/15% methanol solvent system. The elution volume containing both 5-HIAA and HVA (Rt approximately 3 min) is collected. Recoveries (mean +/- SD) for this purification step, which is monitored using fluorometric detection, were usually above 90%. After acidification of the approximately 2 ml collected fraction, 100 microliters is reinjected on a C18 column and separated (Rt: 5-HIAA, 4 min; HVA 5.5 min) using an 80% pH 3.5 phosphate buffer/20% methanol mobile phase. The compounds are determined by flow-through amperometry with absolute detection limits of approximately 25 pg. Both 5-HIAA and HVA are well resolved from other electroactive species present and are easily determined at normal and greatly reduced concentrations in human urine.     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.