染料膜亲和色谱法中膜堆的制备及应用

将染料亲和配基偶联于大孔纤维素膜上,所得亲和膜用胶粘法制成亲和膜堆,膜堆的通透性远优于通常的亲和色谱柱。装有蓝色和红色亲和膜的膜堆可分别用于人血清白蛋白和碱性磷酸酯酶的分离纯化,其中碱性磷酸酯酶可在一步操作后纯化４０倍。     

ProteinA亲合膜色谱柱的性能及从人血浆中纯化免疫球蛋白的研究

以木纤维为基质,与甲基丙烯酸环氧丙酯共聚接枝合成了复合膜介质,用复合膜介质制备了ｐｒｏｔｅｉｎＡ亲合膜色谱柱,考察了ｐｒｏｔｅｉｎＡ亲合膜色谱柱液相流动特性和吸附性能。实验证明：流速与亲合膜色谱柱柱压呈线性关系,当流速为３ｍＬ／ｍｉｎ时,柱压为１６０ｋＰａ。免疫球蛋白（ＩｇＧ）浓度和上样速度是影响ｐｒｏｔｅｉｎＡ键合容量的重要因素,对其进行了优化研究。用动态吸附法确定了对人ＩｇＧ动态最大吸附能力可达２１．７ｍｇ／ｇ（干介质）。     

An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white

This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. The crude egg white extract was passed through a cation exchanger Streamline trade mark SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on dye-linked cellulose beads. Ovalbumin and ovomucoid did not bind to Streamline SP. Ovalbumin could be precipitated from this unbound fraction by 5% trichloroacetic acid, and ovomucoid was removed from the supernatant by precipitation with ethanol. The yields of lysozyme, ovomucoid, and ovalbumin were 77, 94, and 98%, respectively. All the purified proteins showed single bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All the steps are easily scalable, and the process described here can be used for large-scale simultaneous purification of these proteins in the pure form.     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

Determination of phenylethanolamine N-methyltransferase by high-performance liquid chromatography with fluorescence detection

A highly sensitive assay method for phenylethanolamine N-methyltransferase in rat adrenal medulla and brain is described which employs high-performance liquid chromatography with fluorescence detection. Epinephrine formed enzymatically from the substrate norepinephrine and isoproterenol (internal standard), after chromatography on a small cartridge of a cation exchanger, Toyopak SP, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine, a selective fluorescence derivatization reagent for catechol compounds. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for epinephrine formed enzymatically is 0.66 pmol per assay tube.     

分离尿激酶的亲和色谱填料的制备

 合成了分别以 Sepharose和聚甲基丙烯酸环氧丙酯为基质、对氨基苯甲脒为配基的分离尿激酶的两种亲和色谱填料 ,并用于尿激酶粗品的直接纯化 ,活性回收率分别为 1 0 8.3 %和 43 .4% ,比活提高倍数分别为 9.0 6倍和 3 6.9倍。     

Automation of fluorous solid-phase extraction for parallel synthesis

An automatic fluorous solid-phase extraction (F-SPE) technique is developed by using FluoroFlash SPE cartridges on the RapidTrace workstation. A 10-module workstation has the capability to complete a maximum of 100 SPEs each round in 1-2 h. Another important feature of the RapidTrace system is that it has the capability to load slurry samples onto the F-SPE cartridges. The F-SPE cartridge charged with 2 g of fluorous silica gel is used to purify up to 200 mg of crude sample. Sample loading, elution solvent, cartridge reuse, and SPE reproducibility are evaluated. The automatic SPE system is used for purification of a small urea library generated from amine-scavenging reactions using fluorous dichlorotriazine, a 96-membered amide library generated using 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine as the coupling agent, and another 96-membered library generated from fluorous Mitsunobu reactions. Approximately 90% of the products have > 90% purity after F-SPE.     

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.     

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

促卵泡激素的径向色谱纯化工艺

 比较了径向离子交换色谱与经典离子交换色谱在纯化工艺放大过程中的分离效果 ,证实了径向色谱比经典色谱更适合于促卵泡激素 (FSH)的纯化 ,并建立了适合于大规模生产FSH的径向色谱纯化新工艺。纯化后FSH的比活性为 180IU/mg ,活性回收率为 5 6 % ,产品质量符合国家药典规定。     

Protein A tangential flow affinity membrane cartridge for extracorporeal immunoadsorption therapy.

Tangential flow affinity membrane cartridge (TFAMC) is a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high flow rate. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed IgG and little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable.     

Characterization of molecularly imprinted polymers for the extraction of tobacco alkaloids and their metabolites in human urine

Molecularly imprinted polymers (MIPs) are synthetic polymers designed to selectively extract target analytes from complex matrices (including biological matrices). The literature shows that MIPs have a degree of cross-selectivity from analytes within the same class of compounds. A commercially available MIP for tobacco-specific nitrosamines (TSNAs) is designed to be class selective for four TSNA compounds. This study sought to characterize the extent of cross-selectivity of the TSNA MIPs with other tobacco alkaloids. Cross-selectivity and recovery of the SupelMIP™ TSNA SPE cartridges was assessed with N-nitrosonornicotine (NNN), nicotine, cotinine and morphine. Their recoveries were compared with the recoveries of a nonimprinted polymer SPE cartridge, and two traditional SPE cartridges: a Waters mixed-mode cation exchange cartridge and a Waters hydrophilic–lipophilic balance cartridge. NNN and cotinine had the highest recoveries with the MIP cartridge, over 80%, and cotinine samples in urine had >80% recoveries. Nicotine had highly variable recoveries, possibly owing to differing chemical properties from the TSNAs. All three analytes had significantly different recoveries with the MIP cartridges compared with the traditional SPE cartridges. Morphine displayed nonspecific interactions with the MIP cartridges. Utilization of the TSNAs’ cross-selectivity allows for simultaneous extraction and identification of multiple tobacco biomarkers using one extraction technique.     

用新型尼龙亲和膜纯化人血浆蛋白

 以微孔尼龙膜为基质 ,分别采用三氯三嗪和 1,4 丁二醇二甘油醚活化法制备了一系列亲和膜。首次使用平板亲和膜方法经一步操作即从人血浆中纯化得到高纯度的γ 球蛋白、纤溶酶原。电泳分析显示 ,纯化得到的人血浆γ 球蛋白 ,纯度在 83%以上 ;纯化得到的γ 球蛋白和纤溶酶原的纯度均高于市售同类产品 ;另外 ,初步探索了人血浆凝血酶的一步纯化法。该法纯化得到的凝血酶比活为 42 0NIH单位 /mg～ 42 5NIH单位 /mg 。     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer

Solid-phase extraction (SPE) was coupled to ion-trap mass spectrometry to determine clenbuterol in urine. For SPE a cartridge exchanger was used and, after extraction, the eluate was directly introduced into the mass spectrometer. For two types of cartridges, i.e. C18 and polydivinylbenzene (PDVB), the total SPE procedure (including injection of 1 mL urine, washing, and desorption) has been optimised. The total analysis, including SPE, elution, and detection, took 8.5 min with PDVB cartridges, while an analysis time of 11.5 min was obtained with C18 cartridges. A considerable amount of matrix was present after extraction of urine over C18 cartridges, resulting in significant ion suppression. With PDVB cartridges, the matrix was less prominent, and less ion suppression was observed. For single MS, a detection limit (LOD) of about 25 ng/mL was found with PDVB cartridges. With C18 cartridges an LOD of only about 50 ng/mL could be obtained. Applying tandem mass spectrometry (MS/MS) did not lead to an improved LOD due to an interfering compound. However, a considerable improvement in the LOD was obtained with MS3. The selectivity and sensitivity were increased by the combination of efficient fragmentation of clenbuterol and reduction of the noise. Detection limits of 2 and 0.5 ng/mL were obtained with C18 and PDVB cartridges, respectively. The ion suppression was 4 to 45% (concentration range: 250 to 1.0 ng/mL) after extraction of urine using PDVB cartridges, and up to 70% ion suppression was observed using C18 cartridges. With MS4, no further improvement in selectivity and sensitivity was achieved, due to inefficient fragmentation of clenbuterol and no further reduction of noise.     

单分散交联聚甲基丙烯酸环氧丙酯树脂的制备及亲合纯化伴刀豆球蛋白-A

采用一步种子溶胀聚合法制备了颗粒呈单分散的交联聚甲基丙烯酸环氧丙南树脂，对其环氧基的组成比进行了表征，并以氨基葡萄糖为配基，首次制备了纯化粗品伴刀豆球蛋白-A(Ⅲ）(Con-A，Ⅲ)的聚合物基质的高效亲合色谱柱。配基在树脂上的键合量为8．2mg／g，对纯化后的Con-A（Ⅲ）的吸附量为13．4mg／g。使用该亲合色谱介质成功地从粗品Con-A中快速纯化了Con-A，电泳分析显示为一个主要的谱带，纯度从15％提高到95％。     

Efficiency and characteristics of solid-phase (ion-exchange) extraction for removal of Cl- matrix

Certain types of samples contain chloride in concentrations that are too high to accurately determine other anions by ion chromatography without any pretreatment. One of the most widely used approaches for such samples is matrix elimination using disposable cartridges containing a cation-exchange resin in the Ag+ form. The efficiency and characteristics of the commercially available cartridge for Cl- removal were tested by the on-line connection of the cartridge effluent to an inductively coupled plasma mass spectrometer. Displacement efficiency of Ag+ ions and loading capacity of the cartridges were studied at different flow-rates. Significant amounts of silver were detected in the effluent, which were attributed to colloidal AgCl as well as dissolved Ag+ ions. Because silver ions can cause irreversible damage to the analytical column, an Ag cartridge followed by an on line filter (pore size 0.22 microm) and cartridge in the H3O+ form were proposed for improvement of this sample pretreatment technique for chloride removal.     

Rapid and sensitive determination of phenylurea herbicides in water in the presence of their anilines by extraction with a Carbopack cartridge followed by liquid chromatography

A rapid and sensitive method for determining phenylurea herbicides in environmental aqueous samples in the presence of their anilines is described. The water sample is preconcentrated by passage at a flow-rate of ca. 150 ml/min through a 250-mg graphitized carbon black (Carbopack B) cartridge. After washing with 0.6 ml of methanol, the Carbopack B trap is connected with a cartridge containing a strong cation exchanger. Organics trapped by the Carbopack cartridge are eluted by passage of 6 ml of methylene chloride-methanol (95:5, v/v). Anilines and other basic compounds are quantitatively subtracted from the solvent system while flowing through the cation-exchange cartridge. After evaporation and redissolution, the sample is subjected to reversed-phase gradient elution high-performance liquid chromatography with UV detection at 250 nm. Recoveries of phenylureas added to water at levels between 30 and 3000 ng/l were higher than 92%. The limit of detection was about 1 ng/l, for a 2-1 sample. With respect to an octadecyl (C18)-bonded silica cartridge, the Carbopack B cartridge had a far better extraction efficiency for polar phenylureas.     

A magnetizable solid phase for enzyme extraction

A method for the convenient and reliable preparation of magnetizable agarose beads containing iron particles is described. The beads were treated with the triazine dye, Reactive Red 120, and the matrix was examined for the ability to extract proteins from crude preparations using lactate dehydrogenase from porcine muscle as a model. The recovery and specific activity values of enzyme obtained using this matrix and magnetic field separation were significantly greater than those for enzyme purified by centrifugation and conventional dye ligand chromatography.     

Production,purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60 degrees C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0, retaining >80% of its original activity within this range. Half-lives of 150 min at 50 degrees C and 6.5 min at 60 degrees C were found. K(m) and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birchwood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-D-glucuronoxylan with a K(m) of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions as demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Presence of matrix ions could negatively affect the sensitivity and selectivity of liquid chromatography‐tandem mass spectrometer (LC‐MS/MS). In this study, the efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions was demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with LC‐MS. The miniaturized silica monolith with hydroxyl groups present on the largely exposed surface area function as a weak cation exchanger for solid phase extraction (SPE). The miniaturized silica cartridge in 1 cm diameter and 0.5 cm length was housed in a 2‐ml syringe fixed over a SPE vacuum manifold for extraction. The cleaning effectiveness of the cartridge was confirmed by osmometer, atomic absorption spectrometer, LC‐MS and GC‐TOFMS. The drugs were efficiently extracted from urine samples with recoveries ranging from 86% to 114%. The extracted analytes, after concentration and reconstitution, were quantified using LC‐MS/MS. The limits of detection for morphine and codeine were 2 ng/ml and 1 ng/mL, respectively. The relative standard deviations of measurements ranged from 3% to 12%. The monolithic sorbent offered good linearity with correlation coefficients > 0.99, over a concentration range of 50–500 ng/ml. The silica monolithic cartridge was found to be more robust than the particle‐based packed sorbent and also the commercial cartridge with regards to its recyclability and repeated usage with minimal loss in efficiency. Our study demonstrated the efficiency of the miniaturized silica monolith for removal of matrix ions and extraction of drugs of abuse in urinary screening. Copyright © 2011 John Wiley & Sons, Ltd.