尼龙亲和膜的制备及其对木瓜蛋白酶的分离纯化研究

以尼龙膜为基质,键合壳聚糖的方法对尼龙膜进行改性,使膜的非特异性吸附大大降低,并偶联活性染料亲和基 Cibacron Blue F3GA,得到一种新的染料亲和膜.该亲和膜具有良好的色谱性能,对木瓜蛋白酶有较高的吸附量(235.3mg/g),吸附行为满足 Freundlich 吸附模型,pH值和离子强度对吸附量有明显影响.使用该亲和膜对木瓜粉中的木瓜蛋白酶进行分离纯化,纯化倍数可达46.5倍.     

膜色谱技术在生物大分子分离与纯化中的应用

膜色谱是将液相色谱与膜分离融合于一起的新型生化分离技术,具有选择性高、分离速度快、能耗低、易放大等特点,在生物大分子的分离与纯化方面有广阔的应用前景。本文论述了膜色谱的种类、制备方法及作用机理,并介绍了亲和、疏水、离子交换、多级等四种膜色谱在近五年来的应用进展。     

新型分离纯化技术-亲和超滤及其应用

亲和超滤技术是把亲和层析的高选择性和超滤技术的高处理能力相结合的一种新型能大规模进行生物特征物质分离提纯的技术.简要介绍了亲和超滤技术的原理和特征,详细介绍了亲和超滤技术在生物工程和制药工程上的应用.随着亲和超滤技术的发展,应用领域将会不断扩大,将会极大地推动热敏物质(蛋白质、酶、维生素、中草药等)和分子量相近物质(同分异构体、同系物等)分离技术的发展.     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究（Ⅲ）混合酶和蛋清（壳）中的溶菌酶提取及纯化新工艺

采用亚氨二乙酸（IDA)-Cu^2 亲和膜分离新工艺,分离纯化混合酶和蛋壳中的溶菌酶,实验结果表明,亲和膜对溶菌酸的选择吸附性良好,对葡萄糖氧化酶基本不吸附;用亲和膜直接从蛋清（壳）中分离提纯溶菌酶,通过正交实验,找出了最佳操作条件,在此条件下提取的溶菌酶纯度大于17500U/mg.     

氢同位素纯化分离用钯基膜的研究

本文介绍了氢同位素纯化、分离用钯合金膜的制备技术、种类及其应用情况。根据实际应用情况并分析其优缺点,钯基复合膜其综合性能优异成为今后钯基膜的发展方向,同时还要研发机械强度和耐热性更高的膜支撑体及低成本钯基膜的制备方法,探索高使用寿命、高透过率的新型复合膜。     

尼龙亲和膜的制备条件优化及其表征

尼龙膜经稀盐酸水解、壳聚糖改性后,以木瓜蛋白酶为亲和膜的配基,通过戊二醛的活化处理后采用共价结合的方法将配基键合在尼龙膜上,从而得到有特异吸附性能的尼龙亲和膜.本实验考察了制备尼龙亲和膜的交联剂戊二醛的质量分数、pH值、温度、反应时间和酶用量对亲和膜上木瓜蛋白酶活力的影响,确定了最适合的制备条件:pH=9.0,质量分数为0.5%的戊二醛,反应温度为45℃,反应时间为6 h,酶用量为10 mg/mL.该优化条件下制备的尼龙亲和膜具备优良的色谱性能,可用于分离纯化半胱氨酸蛋白酶抑制剂.     

大气中放射性氙的分离纯化系统研制

为实现大气中放射性氙的高效富集,利用制备色谱技术并结合吸附材料性能的差异,开展放射性氙分离纯化技术的研究,通过研究结果确定分离纯化系统的设计参数。实验验证氙分离纯化系统的性能,系统全流程的时间小于1h,获得的氙样品回收率大于90%,纯度高于95%,能满足放射性测量的要求。     

金属螯合膜的制备及其对木瓜蛋白酶的分离纯化

制备了一种能有效分离纯化木瓜蛋白酶的金属螯合膜.该膜以包裹壳聚糖的尼龙膜为基质,通过环氧氯丙烷交联活化、亚氨基二乙酸偶联、金属镍离子螯合制备得到.研究了吸附时间、溶液的pH、溶液的离子强度等因素对吸附的影响,考察了吸附模型,并对其色谱性能和再生性进行探讨.结果表明,最佳吸附条件pH 10.0,反应时间60 min,离子强度0 mol/L,吸附量达67.2 mg/g,吸附行为满足Freundlieh等温吸附模型,其色谱过程中的洗脱剂为1.0 mol/L NaSCN的Tris-HCI(pH5.0)其纯化倍数可以达到19.1倍.吸附洗脱实验重复3次,吸附率仪降低8.7%,表明该模具有良好的稳定性,重复性和再生性,能有效用于木瓜蛋白酶的分离.     

离子液体支撑液膜用于有机物分离的研究进展

离子液体支撑液膜传质速率高,选择性好,稳定性高,在有机物分离中具有独特的优势.总结了离子液体作为膜液相的优点,介绍了离子液体支撑液膜材料的选择,制备以及在有机物分离中的应用,分析了影响离子液体支撑液膜稳定性的因素,展望了其工业化前景.     

电去离子过程脱除低浓度铜离子的研究

采用一级两段的膜堆,以模拟废水为对象,考察了电去离子(EDI)过程脱除低浓度铜离子的性能,为开发一种更环保更经济的重金属废水处理技术提供理论与实验基础.研究证实,脱除铜离子的EDI存在"增强传质"和"电再生"两种模式.在"增强传质"模式操作时,淡室的树脂保持为盐型,阴膜的浓室侧表面无结垢产生.在"电再生"模式下,树脂被水解离产生的H 和OH-所再生,EDI可将铜离子从 50mg/L 左右脱除至火焰原子吸收分光光度法无法检出,同时在阴膜浓室侧表面形成黑色的CuO结垢.选择适当的膜堆形式和工艺条件能够防止结垢,获得一个连续稳定的EDI过程.     

超滤法分离提纯无患子皂苷

采用水提一超滤法分离提纯无患子皂苷.实验考察了絮凝剂的用量,超滤时温度、膜面流速、压力、超滤液pH、膜截留分子量等对分离纯化效果的影响.结果表明在水提液中加入体积分数为2.0%的壳聚糖一醋酸絮凝剂时,预处理效果较好.正交实验表明,采用截留分子量为20 K-50 K的超滤膜,在温度25℃、膜面流速2.78×10-5 m/s、压力0.08 MPa的条件下,所得无患子总皂苷的纯度可达67.02%;而采用6 K超滤膜所得无患子皂苷的产品纯度可达.72.42%.     

Separation and purification of methadone enantiomers by continuous- and interval-flow electrophoresis

Continuous- or free-flow electrophoresis is based upon a thin film of fluid flowing between two parallel plates. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. Using the Octopus apparatus in a horizontal position, continuous preparative separation of methadone enantiomers in the presence of (2-hydroxypropyl)-β-cyclodextrin as a chiral selector was investigated under conditions of continuous-flow zone electrophoresis and continuous-flow isotachophoresis. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary electrophoresis and circular-dichroism spectroscopy. In both electrophoretic modes, partial separation of the two enantiomers with an enrichment of about 80% and a throughput of 10-20 mg of racemic methadone per hour was obtained. Operating the Octopus apparatus with interrupted buffer flow during electrophoresis, a process termed interval-flow electrophoresis, resulted in complete separation of milligram quantities of the two methadone enantiomers. Furthermore, commencing with racemic methadone, continuous multistage isotachophoretic processing is shown to be suitable to purify (R)-(-)-methadone, the enantiomer with higher pharmacological activity, on a mg/h scale and at a mM concentration in the collected product stream.     

Separation and purification of hyaluronic acid by glucuronic acid imprinted microbeads

The purification of hyaluronic acid (HA) is relatively significant to use in biomedical applications. The structure of HA is formed by the repetitive units of glucuronic acid and N-acetyl glucosamine. In this study, glucuronic acid-imprinted microbeads have been supplied for the purification of HA from cell culture (Streptococcus equi). Histidine-functional monomer, methacryloylamidohistidine (MAH) was chosen as the metal-complexing monomer. The glucuronic acid-imprinted poly(ethyleneglycoldimethacrylate-MAH-Copper(II)) [p(EDMA-MAH-Cu²⁺)] microbeads have been synthesized by typical suspension polymerization procedure. The template glucuronic acid has been removed by employing 5 M methanolic KOH solution. p(EDMA-MAH-Cu²⁺) microbeads have been characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) images and swelling studies. Moreover, HA adsorption experiments have been performed in a batch experimental set-up. Purification of HA from cell culture supernatant has been also investigated by determining the hyaluronidase activity using purified HA as substrate. The glucuronic acid imprinted p(EDMA-MAH-Cu²⁺) particles can be used many times with no significant loss in adsorption capacities. Also, the selectivity of prepared molecular imprinted polymers (MIP) has been examined. Results have showed that MIP particles are 19 times more selective for glucuronic acid than N-acetylglucose amine.     

Absolute quantification of human serum transferrin by species-specific isotope dilution laser ablation ICP-MS

We report for the first time the absolute quantification of a metalloprotein separated by nondenaturing gel electrophoresis (GE) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in combination with species-specific isotope dilution mass spectrometry (IDMS). The proposed method is based on the use of an isotopically enriched (57)Fe-transferrin complex to quantify natural transferrin (Tf) in human serum samples. First, the saturation process of Tf with natural abundance or isotopically enriched (57)Fe was accomplished by using freshly synthesized Fe-citrate solutions. The stability of the metal-protein complex as well as its stoichiometry was investigated by spectrophotometry and ICP-MS, demonstrating a satisfactory stability over a period of at least one month and a molar ratio Fe:Tf of 1.94 ± 0.09, which is close to the expected value of 2. The species-specific IDMS method was compared with external calibration using the Fe-Tf (absolute Tf amount between 2 and 10 μg) and different sample preparation procedures (stained and nonstained gels) as well as two laser ablation strategies (single line ablation in the direction perpendicular or horizontal to the electrophoretic migration) were evaluated. The proposed species-specific GE-LA-ICP-IDMS method was tested for the analysis of a serum certified reference material (ERM-DA470k/IFCC). The results were in good agreement with the certified value with relative standard deviation values in the range of 0.9-2.7% depending on the data treatment procedure used. Furthermore, the analysis time has been drastically reduced in comparison with previous approaches to less than 15 min. The quantification by species-specific GE-LA-ICP-IDMS allowed us to obtain accurate and precise results not only by analyzing the protein spot in the middle position but also in the adjacent ablation line to the center.     

乙酸纤维-EGCG分子印迹复合膜分离纯化茶多酚中的EGCG

采用多孔的乙酸纤维膜为支撑体,制备EGCG分子印迹复合膜并将该膜用于分离富集茶多酚中的EGCG.EGCG高效液相色谱检测结果表明,该方法分离获得的EGCG纯度达到93%.     

Separation and quantification of two fluoroquinolones in serum by on-line high-performance immunoaffinity chromatography

To demonstrate that two structurally similar chemicals can be extracted from a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-switching system was used, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolone sarafloxacin was covalently cross-linked to a protein G column and used to capture fluoroquinolones in fortified serum samples. Interference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the injection port and the Mab Sara-95 derivatized HPIAC column. Upon injection, serum samples containing the fluoroquinolones passed through both columns. The cleanup column detained serum components, that otherwise would bind nonspecifically to the HPIAC column, but allowed the fluoroquinolones to pass through unhindered to the HPIAC column. The fluoroquinolones were then eluted from the HPIAC column according to their relative affinities for the antibody, and individual peaks were monitored using fluorescence detection. By using an on-line cleanup column in tandem with an HPIAC column, the fluoroquinolones could be separated from the serum matrix and then separated from each other on the basis of their affinity for Mab Sara-95 without the use of organic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compounds in a complex matrix.     

HPLC-ICPMS and stable isotope-labeled approaches to assess quantitatively Ti(IV) uptake by transferrin in human blood serum

Little is known about the effects of titanium found in patients wearing prostheses or about the biochemical pathways of this metal when used as an anticancer drug (e.g., titanocene dichloride). In this work, transferrin has been confirmed as the only carrier protein binding Ti in human blood serum samples by making use of different HPLC protein separations followed by element-specific Ti detection by ICPMS. Besides, isotope dilution analysis has been applied to the quantitative speciation of Ti-Tf in standards and human blood serum samples. Species-unspecific and species-specific isotope dilution modes have been explored. In the first case, very low Ti-Tf results were obtained even using two different chromatographic mechanisms, anion exchange (20-24%) and size exclusion (33-36%). Surprisingly, no major Ti species except Ti-Tf were observed in the chromatograms, suggesting that Ti(IV) hydrolysis and precipitation as inactive titanium oxide species could take place inside the chromatographic columns. These results demonstrate that chemical degradation of metalloproteins during analytical separations could ruin the sought speciation quantitative results. The isotope dilution species-specific mode, much more accurate in such cases, has been instrumental in demonstrating the possibility of gross errors in final metalloprotein quantification. For this purpose, an isotopically enriched standard of (49)Ti-Tf was synthesized and applied to the quantitative speciation of Ti-Tf again. Using this species-specific spike, Ti-Tf dissociation inside the chromatographic columns used could be corrected, and thus, quantitative Ti-Tf binding in serum (92-102%) was observed. In other words, the usefulness and potential of a species-specific isotope dilution analysis approach to investigate quantitatively metal-protein associations, which can be dissociated at certain experimental conditions, is demonstrated here for the first time.     

Accurate determination of human serum transferrin isoforms: Exploring metal-specific isotope dilution analysis as a quantitative proteomic tool

Carbohydrate-deficient transferrin (CDT) measurements are considered a reliable marker for chronic alcohol consumption, and its use is becoming extensive in forensic medicine. However, CDT is not a single molecular entity but refers to a group of sialic acid-deficient transferrin isoforms from mono- to trisialotransferrin. Thus, the development of methods to analyze accurately and precisely individual transferrin isoforms in biological fluids such as serum is of increasing importance. The present work illustrates the use of ICPMS isotope dilution analysis for the quantification of transferrin isoforms once saturated with iron and separated by anion exchange chromatography (Mono Q 5/50) using a mobile phase consisting of a gradient of ammonium acetate (0-250 mM) in 25 mM Tris-acetic acid (pH 6.5). Species-specific and species-unspecific spikes have been explored. In the first part of the study, the use of postcolumn addition of a solution of 200 ng mL(-1) isotopically enriched iron (57Fe, 95%) in 25 mM sodium citrate/citric acid (pH 4) permitted the quantification of individual sialoforms of transferrin (from S2 to S5) in human serum samples of healthy individuals as well as alcoholic patients. Second, the species-specific spike method was performed by synthesizing an isotopically enriched standard of saturated transferrin (saturated with 57Fe). The characterization of the spike was performed by postcolumn reverse isotope dilution analysis (this is, by postcolumn addition of a solution of 200 ng mL(-1) natural iron in sodium citrate/citric acid of pH 4). Also, the stability of the transferrin spike was tested during one week with negligible species transformation. Finally, the enriched transferrin was used to quantify the individual isoforms in the same serum samples obtaining results comparative to those of postcolumn isotope dilution and to those previously published in the literature, demonstrating the suitability of both strategies for quantitative transferrin isoform determination in real samples.     

Strategies to study human serum transferrin isoforms using integrated liquid chromatography ICPMS, MALDI-TOF, and ESI-Q-TOF detection: application to chronic alcohol abuse

Variations in the distribution of sialoforms of human serum transferrin (Tf) in correlation with pathological states, which are associated with abnormalities in glycosylation, is of great clinical interest. In such studies, the methodologies of analysis are required to be sensitive and selective for observing small variations among isoforms and able to characterize the molecular structure of such forms. Thus, the present work describes, in the first part, the separation of transferrin isoforms, after iron saturation of the protein, by high-performance liquid chromatography (HPLC) and the on-line specific atomic detection of the iron present on each of the separated isoforms by on-line coupling the HPLC system to an inductively coupled plasma mass spectrometer (ICPMS). This allowed low detection levels for the different isoforms (L.D. 0.03 microMTf). After screening of the isoforms containing iron by ICPMS, structural characterization of each isoform can be independently carried out. Thus, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) and electrospray mass spectrometry (ESI-Q-TOF) are compared in the second part of this study. The different atomic and molecular MS methods revealed the presence of elevated carbohydrate-deficient transferrin (CDT) isoforms in human serum samples from chronic alcohol consumption patients. MALDI-TOF appeared to be sensitive to concentration levels of the analytes, and the observed mass accuracy was highly compromised by the protein heterogeneity (peak width at half-maximum approximately 2000 Da for every fraction). On the other hand, ESI-Q-TOF allowed good mass accuracy (m < or = 0.05%) and peak width of 45 Da in the deconvoluted spectra; while ICPMS detection could be preferable for sensitive protein isoforms determinations, ESI-Q-TOF turns out to be an excellent "fingerprinting" technique for alcoholism diagnosis.