IDA型Cu~(2 )-螯合亲和膜色谱法对牛肝过氧化氢酶纯化的研究(英文)

首次采用以改性纤维素为基质、亚氨二乙酸（ＩＤＡ）为取代配基的铜离子螯合膜色谱法对牛肝过氧化氢酶（ＢＬＣ）的分离纯化进行了研究。缓冲液的ｐＨ值对ＢＬＣ与螯合配基的结合影响显著。在选定的色谱条件下，ＢＬＣ粗酶液经ＩＤＡ型Ｃｕ２＋－螯合膜色谱柱一步纯化，比活性平均提高４．７倍，回收率为６７．７％。金属螯合膜色谱柱可用含０．２ｍｏｌ／Ｌ的咪唑或５０ｍｍｏｌ／ＬＥＤＴＡ－１ｍｏｌ／ＬＮａＣｌ的缓冲液再生，反复使用，后者比前者对柱子的再生效果更好。     

HPLC/ECD测定人血小板中5-羟色胺浓度

用ＨＰＬＣ／ＥＣＤ法测定血小板５－ＨＴ,运用 ＷａｔｅｒｓμＢｏｎｄａｐａｋ Ｃ柱,３,４二羟基苄胺（ＤＨ－ＢＡ）作内标,以 ０.０７ ｍｏｌ／Ｌ乙酸钠缓冲液＋乙腈为流动相（２０＋１）,每１Ｌ缓冲液含 ０．０５ ｍｏｌ／Ｌ柠檬酸、２．５ ｍｍｏｌ／Ｌ庚烷磺酸钠、０．１ｍｍｏｌ／Ｌ Ｎａ_２ＥＤＴＡ,流速０．８ ｍＬ／ｍｉｎ。ＤＨＢＡ和 ５－ＨＴ的保留时间分别为５．４和１１．５ ｍｉｎ,线性范围为０．０２５～１ｍｇ／Ｌ（ｒ＝ ０．９９９７）。５－ＨＴ日内 ＲＳＤ低于１．８７％,日间ＲＳＤ低于８．５４％,方法回收率为１００．４％±２．３％。提示本方法快速简便,灵敏准确,适用于临床基础研究。     

Mo（Ⅵ）－PAN与Mo（Ⅵ）－PAR体系的极谱性质对比研究

本文比较了Ｍｏ（Ⅵ）－１－（２－吡啶基偶氮）－２－萘酚（ＰＡＮ）与Ｍｏ（Ⅵ）－４（２－吡啶基偶氮）间苯二酚（ＰＡＲ）的极谱性质，讨论它们在作为吸附波试剂时的优缺点，以求得在选择络合剂时的感性和理性认识．本文还报道了Ｍｏ（Ⅵ）－ＰＡＮ－ＫＢｒＯ３吸附催化波体系，最佳实验条件，０．１ｍｏｌ／ＬＨＡｃ－－ＮａＡｃ，ｐＨ＝４．６，０．０１ｍｏｌ／ＬＫＢｒＯ３，２．５×１０－５ｍｏｌ／ＬＰＡＮ．峰电位为－０．７１Ｖ（ｖｓ．ＳＣＥ），检出限１×１０－９ｍｏｌ／Ｌ，线性范围０～６×１０－７ｍｏｌ／Ｌ     

铟（Ⅲ）—2—硫基苯骈噻唑—7—碘—8—羟基喹啉—5—磺酸三元络合物吸附波研究

在ＨＣｌ－ＮａＡｃ介质（ｐＨ２．５）中，用单扫示波极谱法可获得高灵敏的Ｉｎ（Ⅲ）－２－巯基苯骈噻唑（ＭＢＴ）－７－碘－８－羟基喹啉－５－磺酸（Ｆｅｒｒｏｎ）三元络合物吸附波，峰电位在一－０．６４Ｖ（ｖｓ．ＳＣＥ），峰电流与铟浓度在７．０×１０＾－１０ｍｏｌ／Ｌ～８．８×１０＾－７ｍｏｌ／Ｌ之间呈线性关系，检出限可达３．０×１０＾－１０ｍｏｌ／Ｌ，测得电活性络合物的组成为Ｉｎ（Ⅲ）：ＭＢＴ：１∶１∶     

大量锆存在下铪镧钼杂多酸—耐尔蓝体系测定给

本文研究了耐尔蓝（ＮＢ）－给镧钼杂多酸（ＨｆＬａＭｏ－聚乙烯醇（ＰＶＡ）体系测定铪的超高灵敏光度法。在ＰＶＡ存在下，铪，镧和钼酸铵形成杂多酸，继而与耐尔蓝形成离子缔合物。其适宜条件为〔ＨＣｌｏ４〕＝１．２ｍｏｌ／Ｌ，〔Ｌａ×３＋〕＝２．９×１０＾－７ｍｏｌ／Ｌ，〔ＭｏＯ４×２－〕＝１．１×１０＾－３ｍｏｌ／Ｌ，〔ＮＢ〕＝２．７×１０＾－６ｍｏｌ／Ｌ，ＰＶＡ０．０８％。离子缔合物的最大吸收波长在５９     

铬（Ⅵ）－十六烷基三甲基溴化铵极谱吸附波研究

在ｐＨ９．３的Ｎａ２Ｂ４Ｏ７Ｎａ２ＣＯ３缓冲溶液中，铬（Ⅵ）与十六烷基三甲基溴化铵（ＣＴＭＡＢ）在滴汞电极上吸附还原，峰电位－０．３１Ｖ（ｖｓ．ＳＣＥ）．铬（Ⅵ）在１．９×１０－８２．１×１０－６ｍｏｌ／Ｌ范围内与峰高呈线性关系检出限为７７×１０－９ｍｏｌ／Ｌ用于工业废水中铬的测定，结果与光度法一致     

钛(Ⅳ)-5-Br-PADAP-NaClO_3体系极谱吸附催化波的研究

２－（５－溴－２－吡啶偶氮）－５－二乙氨基苯酚（５－Ｂｒ－ＰＡＤＡＰ）在极谱和伏安分析中的应用已有一些报道。陈林林等曾经研究过Ｔｉ（Ⅳ）－５－Ｂｒ－ＰＡＤＡＰ络合物在稀Ｈ２ＳＯ４－Ｈ２Ｃ２Ｏ４溶液中的极谱行为,但灵敏度不高（１．０×１０－７ｍｏｌ／Ｌ）。我们发现,在ｐＨ４．１左右的ＨＡｃ－ＮａＡｃ底液中,Ｔｉ（Ⅳ）－５－Ｂｒ－ＰＡＤＡＰ－ＮａＣｌＯ３体系产生一灵敏的极谱吸附催化波,二次导数峰电位在－０．８６Ｖ（ｖｓ．ＳＣＥ）左右,波形良好,波高稳定,检测限可达４×１０－１０ｍｏｌ／Ｌ。１　实验部…     

磷酸三丁酯萃淋树脂吸附铬(Ⅵ)的性能及机理

研究了磷酸三丁酯萃淋树脂吸附的性能及机理。在３－５ｍｏｌ／Ｌ ＨＣｌ介质中,Ｃｒ以「Ｃｒ２Ｏ＾２－７（ＴＢＰＨ＾＋）形式被吸附,吸附反应在３－５ｍｉｎ内即可达到平衡,其ΔＨ＝－４６．０８ｋＪ／ｍｏｌ饱和吸附容量为２５ｍｇ／ｇ。用减压微色谱柱技术,吸附在柱上的Ｃｒ仅用１ｍＬ水可完全洗脱。此法用于电镀废水中Ｃｒ的测定获得了满意的结果。     

壳聚糖修饰石墨电极半微分伏安法测定L-抗坏血酸

采用滴涂法制备了壳聚糖修饰石墨圆盘电极,并用于Ｌ－抗坏血酸（ＬＡＡ）的半微分伏安法测定。研究了电解液种类及酸度、膜厚度、富集电位及时间、扫速和温度等因素对伏安曲线的影响,获得了较为优化的测试条件。在０．２０ｍｏｌ／Ｌ　ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４（ｐＨ６．０）缓冲液中,ＬＡＡ的浓度在 １． ０×１０－５～ ６． ０ × １０－４ ｍｏｌ／Ｌ范围内与氧化峰电流呈良好线性关系（ ｒ＝０．９９９７）,检测限达６．７×１０－７ｍｏｌ／Ｌ;生物体内常见的干扰物质如亚硝酸盐、多巴胺、葡萄糖等不干扰测定。 ９次测得电极在含 ５．０ ×１０－５ ｍｏｌ／Ｌ ＬＡＡ试液中峰电流的 ＲＳＤ为３．６％,回收率为９６．４％～１０２．８％（ ｎ＝５）。已成功用于维生素Ｃ片剂和人血清中 ＬＡＡ的测定。     

氯化血红素模拟过氧化物酶高效液相色谱柱后衍生法测定过氧化氢和水溶性有机过氧化物

研究了氯化血红素（Ｈｅｍｉｎ）模拟辣根过氧化物酶（ＨＲＰ），对羟基苯乙酸（ＰＨＰＡＡ）作 底物，高效液相色谱（ＨＰＬＣ）柱后衍生荧光法测定Ｈ２Ｏ２和水溶性有机过氧化物的方法。采用 Ｈｅｍｉｎ作催化剂柱后衍生反应的最佳ｐＨ值约为１１，与荧光检测ｐＨ值一致，使得以往ＨＰＬＣ 柱后衍生方法所需的高压泵从３台减少到２台，而一些不能作为ＨＲＰ底物的羟烷基过氧化 物在ｐＨ值≥１０的溶液中迅速水解为Ｈ２Ｏ２从而得到测定。优化了测定Ｈ２Ｏ２和甲基过氧化 氢（ＣＨ３ ＯＯＨ， ＭＨＰ）的条件。最佳条件下 Ｈｅｍｉｎ方法测定 Ｈ２Ｏ２的检测限为 ９．０ × １０－９ ｍｏｌ／Ｌ， 测定 ＭＨＰ的检测限为 ２．０ × １０－７ ｍｏｌ／Ｌ。     

Immobilized metal affinity composite membrane based on cellulose for separation of biopolymers

Ｕｐｔｏｄａｔｅ,ｒａｐｉｄｐｕｒｉｆｉｃａｔｉｏｎｏｆｍｉｘｅｄｐｒｏｔｅｉｎｉｎｌａｒｇｅｓｃａｌｅｈａｓｂｅｅｎａｎｉｍｐｏｒｔａｎｔｒｅｓｅａｒｃｈｐｒｏｊｅｃｔｉｎｂｉｏｅｎｇｉｎｅｅｒｉｎｇｐｒｏｄｕｃｔｐｒｏｃｅｓｓｉｎｇ.Ｉｍｍｏｂｉｌｉｚｅｄｍｅｔａｌｉｏｎａｆｆｉｎｉｔｙｃｈｒｏｍａｔｏｇｒａｐｈｙ(ＩＭＡＣ)ｉｓａｎｅｆｆｉｃｉｅｎｔｍｅｔｈｏｄｅｘｔｅｎｓｉｖｅｌｙｕｓｅｄｆｏｒａｆｆｉｎｉｔｙｐｕｒｉｆｉｃａｔｉｏｎｏｆｂｉｏｌｏｇｉｃａｌｌｙａｃｔｉｖｅｓｕｂｓｔａｎｃｅ…     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究(Ⅱ)──不同金属螯合亲和膜对溶菌酶的吸附性能

采用低温氧或氨等离子体法改性聚丙烯微孔膜,基于等离子体改性膜的活化、偶联及螯合过程的机理,制备了Fe³⁺,Ni²⁺,Cu²⁺和Zn²⁺等金属离子螯合亲和膜,并用于溶菌酶的吸-脱附实验.实验结果表明,Ni²⁺和Cu²⁺离子螯合亲和膜对溶菌酶具有较高的吸附量,螯合过程中采用氯化物盐溶液制得的膜对溶菌酶吸附量比采用硫酸盐溶液制得的膜的吸附量高.两种膜的重复吸-脱附性能相近,而Fe³⁺螯合亲和膜基本上不能用于重复吸-脱附实验.采用补充金属螯合离子,能部分恢复亲和膜对溶菌酶的吸附量,是实现亲和膜重复使用的有效方法.     

金属离子与氨羧类螯合配体的键合特性研究

利用前沿色谱法,通过Cu2+、Ni2+和Co2+与螯合配体IDA 键合的准确度(R2>0.98)与精密度(RSD <5%)实验,研究了前沿色谱法同时测定络合稳定常数KML与总键合位点数Λ0值的可行性.为了进一步证明前沿色谱法的普适性,以Cu2+、Ni2+和Co2+为代表,在3种键合缓冲体系(NaAc-HAc、Na-PB、Tris-HCl)中,考察了金属离子在3种氨羧类螯合配体(IDA、Asp、Glu)上络合稳定常数KML的变化规律.结果表明,螯合配体对金属离子键合强度遵循IDA>Asp>Glu;金属离子对螯合配体键合强度遵循Cu2+>Ni2+>Co2+;3种键合缓冲体系中,NaAc-HAc键合效果最好.按照M06/6-311++G (d, p) 方法对螯合配体与金属离子的结合能(ΔE)与吉布斯自由能(ΔG)进行相关的量子计算.根据ΔE 与ΔG 的大小,从理论上推测出螯合配体与金属离子的键合规律,此规律与上述实验结果基本相符.本研究为金属离子与螯合配体间键合参数的求取提供了有效的方法和手段,从而为今后提高IMAC柱的稳定性,解决固定金属亲和柱在应用过程中尤其是竞争洗脱过程中金属离子的流失问题奠定了良好的基础.     

Covalent conjugation of tetrameric bovine liver catalase to liposome membranes for stabilization of the enzyme tertiary and quaternary structures

Tetrameric bovine liver catalase (BLC) is unstable because of its dissociation into subunits at low enzyme concentrations and the conformational change of the subunits at high temperatures. In this work, for stabilization of BLC, the enzyme was covalently conjugated with liposome membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-glutaryl (NGPE). The NGPE, which was responsible for the BLC/membrane coupling, was altered from 0.05 to 0.2 in its liposomal mole fraction f_G. The catalase-conjugated liposome (CCL) with f_G of 0.15 showed the maximum number of the conjugated BLC molecules of 28 per liposome. The reactivity of CCLs to H₂O₂ was as high as that of free BLC at 25 °C in Tris–HCl buffer of pH 7.4. Among the CCLs, the catalyst with f_G of 0.15 was the most stable at 55 °C in its enzyme activity in the buffer because the appropriate number of BLC/liposome covalent bonding prevented the dissociation-induced enzyme deactivation. Furthermore, the CCL showed much higher stability at 55 °C than the free BLC/enzyme-free liposome mixture and free BLC at the low BLC concentration of 340 ng/mL. This was because BLC in the CCL was located in the vicinity of the host membrane regardless of the catalyst concentration, which could induce the effective stabilization effect of the membrane on the enzyme tertiary structure as indicated by the intrinsic tryptophan fluorescence analysis. The results obtained demonstrate the high structural stability of BLC in the CCL system, which was derived from the covalent bonding and interaction between BLC and liposomes.     

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.     

Isolation of ellagic acid from pomegranate peel extract by hydrophobic interaction chromatography using graphene oxide grafted cotton fiber adsorbent

Ellagic acid, a natural polyphenol, was isolated from pomegranate peel extract by hydrophobic interaction using graphene oxide grafted cotton fiber as a stationary adsorbent. The grafted graphene oxide moieties served as hydrophobic interaction‐binding sites for ellagic acid adsorption. The graphene oxide grafted cotton fiber was made into a membrane‐like sheet in order to complete ellagic acid purification by using a binding–elution mode. The effects of operational parameters, such as the composition of the binding buffer/elution buffer, buffer pH, and buffer concentration, on the isolation process were investigated. It was found that 5 mmol/L sodium carbonate aqueous solution is a proper‐binding buffer, and sodium hydroxide aqueous solution ranging from 0.04 to 0.06 mol/L is a suitable elution solution for ellagic acid purification. Under the optimized condition, the purity of ellagic acid increased significantly from 7.5% in the crude extract to 75.0–80.0%. The pH value was found to be a key parameter that determines the adsorption and desorption of ellagic acid. No organic solvent is involved in the entire purification process. Thus, a simple and environmentally friendly method is established for ellagic acid purification using a graphene oxide‐modified biodegradable and bio‐sourced fibrous adsorbent.     

Kinetics and Thermodynamics of the Adsorption of Copper(Ⅱ) onto a New Fe-Si Adsorbent

A new kind of Fe-Si adsorbent was synthesized by iron oxide and diatomite after calcining and hydrothermal process. The influences of the initial Cu2+ concentration, p H and adsorption time on the Cu2+ removal efficiency were discussed. Three adsorption empirical kinetics equations and two thermodynamics equations were used to simulate the adsorption process. The microstructures of newly developed copper removal materials and properties of copper removal are characterized in details by SEM and EDS. Adsorption mechanism of the adsorbent was discussed. The suitable p H value for Cu2+ removal is 5.0 to 6.0 and the adsorption capacity increases with increasing the initial Cu2+ concentration. The adsorption kinetics of the adsorbent could be better described by pseudo second order kinetic model, whereas the adsorption isotherms highly conform to the Freundlich equation. The main crystalline phase of the adsorbent is Fe(Si O3) which can build porous structures conducive to the Cu2+ adsorption.     

新型侧链液晶聚醚螯合树脂的合成及其对水中Cu2 +的吸附性能研究

以环氧环己烷作为改性单体,通过环氧氯丙烷与环氧环己烷阳离子开环聚合获得分子量约为3000的共聚醚主链。然后以联苯为介晶基元、亚氨基二乙酸为末端螯合基团,成功合成了一种新型结构的侧链液晶聚醚螯合树脂。在对其结构鉴定以及基础物理性质测定的基础上,系统地研究了该树脂对水中Cu²⁺吸附的影响因素、再生性能、吸附选择性、吸附模型以及吸附动力学。结果表明,该树脂对Cu²⁺具有良好的吸附性能、再生性能和选择性,其对水中Cu²⁺的吸附为Langmuir单分子层吸附,吸附过程符合准二级动力学模型。     

Metal-Chelating Nanopolymers for Antibody Purification from Human Plasma

The purification of immunoglobulin G (IgG) from human plasma was performed by using a novel metal-chelated adsorbent with nano size. The non-porous nanoparticles were produced by surfactant free emulsion polymerization of ethylene glycol dimethacrylate (EDMA) and 2-methacryloylamidohistidine (MAH). Then, Cu(II) ions were chelated on the nanoparticles. The nano-poly(EDMA-MAH) nanoparticles were characterized by Fourier transform infrared, scanning electron microscope, atomic force microscope and elemental analysis. The non-porous nanoparticles were spherical form and have 100?C250?nm size distribution. The maximum IgG adsorption capacity of the Cu(II) chelated nanoparticles was found to be 463?mg/g polymer at pH 7.0 in HEPES buffer. Desorption of IgG was performed by 1.0?M NaCl and desorption rate was found to be 97?%. IgG was obtained from human plasma with purity of 94?% (up to 578?mg/g polymer). The non-porous nanoparticles allowed one-step purification of IgG from human plasma.