柱亲和介质用于内毒素去除的研究

以琼脂糖凝胶为基质,采用不同方法活化后,键合多粘菌素B配基,制备了3种用于内毒素去除的亲和介质。亲和介质的配基键合量及对内毒素的去除率随间隔臂长度的增加而增加。考察了盐浓度、pH、温度及流速对亲和介质3去除内毒素的影响。结果表明,亲和介质3在NaC l浓度为0.05~0.5 mol/L,pH为6~10,温度为25~55℃,流速为0.20~0.80 mL/m in范围内对内毒素去除效果最佳,去除率大于90%。     

新型镍离子亲和色谱介质的制备及其性能考察

以氯甲基化聚苯乙烯交联微球(氯球)为载体基质、N,N-二甲基甲酰胺(DMF)为溶剂、N,N-二异丙基乙胺(DIEA)催化,将合成的Lys(Boc)-OEt、氯乙酸乙酯连接到微球上。经水解使羧基裸露,与Ni²⁺螯合,制得新型镍离子亲和色谱介质。将其应用于His-Lys-Tyr三肽及Phe-His-Thr三肽纯化研究。以不含组氨酸的六肽作为杂质模型,用所制备的镍离子亲和介质进行分离。结果表明,所制备的镍离子亲和介质对两个含组氨酸的三肽具有很好的亲和作用,并实现了与不含组氨酸六肽的分离。     

亲和色谱-氢化物发生原子荧光分光光度法纯化检测人血浆中的硒蛋白-P

开发了两步亲和色谱法:肝素-琼脂糖凝胶、Ni-琼脂糖凝胶色谱纯化人血浆中硒蛋白-P的方法,并采用氢化物发生-原子荧光分光光度法(HG-AFS)检测,成功搭建了硒蛋白-P的纯化检测平台。确定了亲和色谱纯化的最佳梯度洗脱条件,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)定性检测,得到了一定纯度的硒蛋白-P,其回收率达43.2%。HG-AFS方法的线性相关系数为0.999 1,检出限为0.09μg/L,日内精密度(RSD)为0.12%,日间精密度(RSD)为0.27%,加标回收率为95%～104%。该亲和色谱纯化方法简单易控、回收率高,HG-AFS检测灵敏度高,结果准确可靠。     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究(Ⅰ)──低温氧等离子体改性条件对亲和膜结构与吸附性能的影响

用低温氧等离子体方法对聚丙烯微孔膜表面进行改性.扫描电镜、红外光谱和光电子能谱综合分析结果表明,膜表面上产生了-OH、-COOH和C=O等极性基团,这些基团可被活化和偶联制亲和膜.所制备的Cu²⁺离子金属螯合亲和膜用于对溶菌酶的吸附,在20min和68W的最佳条件下,制成的亲和膜对溶菌酶的吸附量为8μg/cm².     

超大孔离子交换制备色谱分离纯化高活性凝血因子VIII

建立了一条从人血浆中分离高活性凝血因子VIII(FVIII)的纯化工艺。基于FVIII和介质孔径的尺度比及其对蛋白质活性影响的分析,设计了以超大孔离子交换制备色谱为核心步骤的新型分离纯化工艺。分别进行超大孔离子交换色谱与传统离子交换色谱的条件优化,并对优化工艺所得产品进行了活性检测(底物显色法)和纯度检测(高效凝胶过滤和凝胶电泳)。结果表明,超大孔介质结构不但可以有效地保护蛋白质大分子结构,而且能够大幅度地提高制备色谱的传质速率,从而得到具有高凝血活性的FVIII产品。FVIII在超大孔制备色谱过程中的回收率(85%)比传统离子交换制备色谱高4～5倍,产品比活高达154 IU/mg。此外,还研究了超大孔介质的再生程序,采用5个柱体积的1 mol/L NaOH低流速清洗色谱柱,保证了色谱工艺的稳定性。本纯化工艺步骤简单,重现性好,易于放大生产。     

以寡聚组氨酸为配基的高效亲和色谱研究

亲和色谱在所有色谱分离中选择性最高.以组氨酸为配基的亲和介质(Affinitymedia)对免疫球蛋白IgG具有亲和力,显示了良好的分离效果[1];以组氨酸为配基的亲和色谱分离纯化IgG的机理[2]以及组氨酸亲和色谱分离IgG亚基也已有报道[3].组氨酸配基亲和色谱法具有简单、有效和价廉等特点,在免疫化学、临床分析及血液制品的生产等方面均有良好的应用前景.本文分别以含1,3,5个组氨酸残基的小肽为配基,合成了系列亲和介质,并对其亲和色谱行为进行了评价,研究了不同链长的寡聚组氨酸与IgG的亲和相互作用.1　实验部分1.1　固相肽合成　采用Fmoc肽合…     

Cu/Hβ催化剂NH3选择性催化还原NO性能研究

采用浸渍法制备了以Hβ分子筛为载体的负载氧化铜催化剂,考察了Cu负载量对催化剂NH₃选择性催化还原NO反应(NH₃-SCR)性能的影响,通过XRD、N₂吸附-脱附、NH₃-TPD、NO-TPD、H₂-TPR、EDS和XPS等表征技术研究了催化剂的物理化学性质和SO₂存在条件下催化剂活性降低的原因。结果表明,反应气体不含SO₂,Cu负载量为3%,即Cu(3)/Hβ催化剂有较高的反应活性,t₉₅为169℃;反应气体含SO₂,Cu负载量为2%时,即Cu(2)/Hβ催化剂的反应活性较好,t₉₅为225℃。反应前后催化剂的分析结果表明,SO₂存在条件下催化剂活性降低的主要原因是在低温条件下,SO₂与NH₃反应生成的硫铵盐覆盖了催化剂活性中心。     

嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化

抗体药物在癌症治疗和免疫诊断中起着重要作用，但抗体的分离纯化通常采用酸性洗脱，易导致抗体聚集失活等问题。本研究以硅胶为基质，以温敏嵌段聚合物聚[(N-异丙基丙烯酰胺)-b-4-乙烯基吡啶](P[NIPAM-b-4VP])为间隔臂，以4-巯基乙基吡啶(MEP)为配基，制备了一种嵌段共聚温敏亲和色谱固定相SiO₂-P[NIPAM-b-4VP]-MEP,并以牛血清白蛋白(BSA)和γ-球蛋白为模型蛋白，对制备的温敏亲和色谱固定相的色谱性能进行了表征。分别考察了流动相盐浓度和温度对二者分离性能的影响。结果表明，在40℃时该固定相只选择性保留γ-球蛋白，而不保留BSA;在5℃时采用含0.6 mol/L NaCl的Tris-HCl(pH 8.0)缓冲溶液进行洗脱，γ-球蛋白的质量回收率为92.7%。该固定相对γ-球蛋白的吸附量为(71.5±2.1) mg/g(n=3),是传统温敏亲和色谱固定相SiO₂-PNIPAM-MEP的2倍。将该固定相用于人血清中IgG的分离纯化，仅通过改变流动相温度和盐浓度即可一步实现对抗体的分离纯化，纯度大于97.4%±0.7%...     

利用高度结构对称的共聚试剂制备高比表面积的硼亲和整体柱

硼亲和整体柱是一种能对顺式二羟基化合物进行选择性分离的重要色谱介质。然而,整体柱微结构在制备过程中难以控制。本文以分子结构高度对称的三聚氰胺和三(2,3-环氧基丙基)异氰酸酯(TEPIC)作为共聚试剂,利用“团队硼亲和”原理,采用原位聚合的方法制备了一种新型硼亲和整体柱。“团队硼亲和”的作用使该硼亲和整体柱对顺式二羟基化合物的结合pH降低至中性范围。结构高度对称的共聚试剂有效地提高了整体柱材料的比表面积,使其达80.3 m²/g,显著高于文献中报道的其它硼亲和整体柱。     

利用高度结构对称的共聚试剂制备高比表面积的硼亲和整体柱

硼亲和整体柱是一种能对顺式二羟基化合物进行选择性分离的重要色谱介质。然而,整体柱微结构在制备过程中难以控制。本文以分子结构高度对称的三聚氰胺和三(2,3-环氧基丙基)异氰酸酯(TEPIC)作为共聚试剂,利用“团队硼亲和”原理,采用原位聚合的方法制备了一种新型硼亲和整体柱。“团队硼亲和”的作用使该硼亲和整体柱对顺式二羟基化合物的结合pH降低至中性范围。结构高度对称的共聚试剂有效地提高了整体柱材料的比表面积,使其达80.3 m²/g,显著高于文献中报道的其它硼亲和整体柱。     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

水热法制备具有高光催化活性的镧掺杂二氧化钛纳米晶

采用水热法合成钛酸钾(K₂Ti₈O₁₇)纳米棒,并将它作为前驱体水热转晶合成TiO₂纳米晶,同时通过在水热体系中引入稀土元素La³⁺实现对TiO₂的La掺杂.考察了不同条件下钛酸盐向TiO₂的转晶过程,发现水热溶液的pH值、温度以及预处理步骤对转晶过程有很大的影响.利用X射线衍射以及透射电子显微镜对样品的晶相和形貌进行了表征.利用电感耦合等离子体原子发射光谱测量了所合成的La掺杂TiO₂样品中的La含量.通过在紫外光下降解甲基橙(MO,10mg/L)测试了La掺杂TiO₂样品的光催化性能.结果表明La掺杂后TiO₂的光催化活性大大提高.在0.15mol/LLa³⁺浓度下180oC水热合成的La掺杂TiO₂样品显示了最佳的光催化活性.其对MO的光催化降解反应常数高达0.11min^-1,大约是空白TiO₂样品的9.20倍,P25TiO₂的3.69倍.     

Immobilization of manganese peroxidase fromLentinula edodes on azlactone-functional polymers and generation of Mn3+ by the enzyme-polymer complex

Manganese peroxidase (MnP) purified fromLentinula edodes was covalently immobilized on 3M’s azlactone-functional copolymer, 3M EmphazeTM AB1 Biosupport Medium. Tethered MnP is capable of generating Mn³⁺ from Mn²⁺ and H₂O₂. Mn₃₊, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn³⁺ -generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn²⁺ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations < 2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn²⁺. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40–50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies > 65% with 100% of the coupled enzyme active have been measured.     

Immobilization of the restriction endonuclease PstI

PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity. It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible for inactive enzyme preparations.     

Refolding of hexahistidine-tagged organophophorous hydrolase from inclusion bodies

The inclusion bodies of organophosphorous hydrolase hexahistidine-tagged at the N-terminus of the protein molecule were isolated from E. coli DH5a cells and purified. The optimum conditions for the solubilization of the inclusion bodies are the following: 6M urea in a phosphate-salt buffer with pH 7.6, 37°C, 2 h. The refolding of the enzyme from solutions of the solubilized inclusion bodies was carried out using metal-chelating affinity chromatography. The activation of the refolded enzyme was studied. The highest catalytic activity of the enzyme is observed after 24-h-long incubation at 4°C in a solution containing 0.05 M CO ₃ ^2? and 10^?5 M Co²⁺.     

Lipase bound cellulose nanofibrous membrane via Cibacron Blue F3GA affinity ligand

Cellulose (Cell) nanofibrous membranes were prepared by nucleophilic reaction of the cellulose hydroxyl with the triazinyl chloride of Cibacron Blue F3GA (CB) ligand and studied as affinity membranes for lipase enzyme. Cell nanofibrous membranes containing fibers with 200 nm average diameters were prepared by electrospinning of cellulose acetate (CA), followed by alkaline hydrolysis. The CB capacity of the Cell nanofibrous membranes was optimized by lengthening the nucleophilic reaction time and increasing CB concentration and ionic strength. The equilibrium adsorption isotherms of CB on the Cell nanofibrous membranes followed a typical Langmuir monolayer adsorption behavior. At 242 mg CB/g of Cell, the maximum lipase adsorption capacity (q_m) and the dissociation constant (K_d) values were 41.02 mg/g and 0.25 mg/mL, respectively. Optimal lipase adsorption capacity was obtained at pH 4.0, its isoelectric point, with added NaCl on Cell membranes 86 mg CB capacity per g of Cell. A facile lipase loading capacity of 16.21 mg/g of CB–Cell was achieved under moderated conditions and could be optimized to reach at least 150 mg/g. The CB–Cell bound lipase had similar catalytic rate and retained 86.2% activity as in its free form. These findings clearly show that the CB bound Cell nanofibrous membrane is a highly efficient ultra-high specific porous support for lipase enzyme and is potentially versatile for immobilizing other enzymes and as affinity membrane for proteins.     

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli

An efficient affinity‐purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4‐Aminopyrrole‐2‐carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C₈ column and reducing SDS‐PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS‐PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ～46 kDa. The desorption constant K_d and theoretical maximum absorption Q_max were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.     

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH₄OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin–TBA complex and 1 M NH₄OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin–TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein–DNA complexes.

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Speciation analysis of orthophosphate and myo‐inositol hexakisphosphate in soil‐ and plant‐related samples by high‐performance ion chromatography combined with inductively coupled plasma mass spectrometry

A novel method based on high‐performance ion chromatography inductively coupled plasma mass spectrometry employing strong anion exchange chromatography with HNO₃ gradient elution for simultaneous analysis of orthophosphate and myo‐inositol hexakisphosphate (IP₆) in soil solution and plant extracts is presented. As inductively coupled plasma mass spectrometry analysis of phosphorus at m/z 31 is hampered by N‐based interferences, ³¹P was measured as ³¹P¹⁶O⁺ at m/z 47 employing dynamic reaction cell technique with O₂ as reaction gas. Orthophosphate and IP₆ were separated within a total chromatographic run‐time of 12 min revealing a limit of detection of 0.3 μmol/L. The coefficients of determination obtained in a working range of 1–100 and 1–30 μmol/L were 0.9991 for orthophosphate and 0.9968 for IP₆, respectively. The method was successfully applied to extracts from three different soils as well as root and shoot extracts of Brassica napus L. The precision of three independently prepared soil extracts was in the range of 4–10% relative standard deviation for PO₄^3? and 3–8% relative standard deviation for IP₆. Soil adsorption/desorption kinetics for IP₆/orthophosphate were performed for investigating the sorption behavior of the two P species in the experimental soils.     

Substrate Related O,O-Dialkyldipeptidylaminophosphonates,A New Type of Thrombin Inhibitor

Abstract

The facile reduction of O,O-dialkyl 1-hydroxyiminoalkanephosphonate precursors, using LiBH₄/Me₃SiCl in THF at ambient temperature, conveniently affords O,O-dialkyl 1-aminoalkanephosphonates in good yield and high state of purity. O,O-Dialkyl 1-aminobenzylphosphonates may be prepared in high yield and purity from catalytic hydrogenolysis of their 1-benzylaminobenzylphosphonate precursors. These biologically active aminophosphonates, when coupled to substrate derived dipeptides, produced a range of novel phosphonotripeptides based upon the ‘fibrinogen-like’ sequence H-D-Phe-Pro-Arg; where the phosphorus structural units replace the ‘Pl’Arg. These tripeptides showed a marked inhibitory specificity towards the trypsin-like serine protease thrombin, a ubiquitous enzyme that plays a crucial role in the cardiovascular system. The compounds possess an initial K_i in-vitro in the micromolar range against thrombin. Further enzyme kinetic analysis of the compound Z-D-Dpa-Pro-Pgl^P(OiPr)₂ (IC₅₀ 11.7 micromolar), showed that it displayed competitive inhibition characteristics toward thrombin, in contrast to the two stage slow-tight binding kinetics that had been shown by the analogous O,O-diphenyl derivative.