染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究

采用反相悬浮交联法制备壳聚糖微球,对微球进行羟丙基氯化及氨基化,并偶联色素配体Cibacron Blue F3GA,得到一种新型染料亲和吸附剂.以牛血清白蛋白(BSA)为目标蛋白,考察了该染料亲和吸附剂的吸附性能,发现其对BSA有较高的吸附量(95.2mg/g),吸附行为满足Langmuir吸附等温式.负载牛血清白蛋白的微球容易洗脱,洗脱率高达99％.     

新型亲和膜色谱用于去除胆红素的研究

 以纤维素膜为亲和基质 ,接枝后分别以聚赖氨酸和季铵盐为配基 ,制备了两种亲和膜介质 ,用于去除磷酸缓冲液 (pH 7 4)及人血白蛋白 (HSA)溶液中的胆红素。实验结果表明 ,该方法对磷酸缓冲液中胆红素的去除率能达到 70 %以上 ;对HSA溶液中的胆红素 ,其去除率稍低 ,但在较低浓度的HSA溶液中 ,也能获得高于 5 0 %的去除率。并对亲和膜与胆红素间的亲和作用进行了研究 ,考察了温度、流速、HSA浓度及胆红素初始浓度等相关条件对去除效果的影响。结果表明 ,在较高的温度条件下 ,去除效果较佳 ;在较高的初始浓度情况下 ,胆红素的吸附速率较大。     

壳聚糖亲和磁性毫微粒的制备及其对蛋白质的吸附性能研究

以壳聚糖为包裹材料包埋自制的磁流体 ,制备了具有核 壳结构的磁性毫微粒 ,并偶联色素配基CibacronBlue 3GA(偶联量 1 4 .5μmol/mL)得到了一种新型亲和磁性毫微粒 .结果表明 ,所得亲和磁性微球具有较窄的粒径分布、形状规整 .以牛血清白蛋白 (BSA)和溶菌酶 (Lys)为目标蛋白 ,考察了该亲和磁性毫微粒的吸附性能 ,发现其对BSA和Lys的吸附量分别为 4和 2 8mg/g,吸附行为满足Langmuir吸附等温式 ,且对时间依赖性小而对溶液离子强度敏感 .     

茜素红与蛋白质分子Langmuir吸附聚集反应研究

在pH4.3的B-R缓冲体系中,用微相吸附-光谱修正技术[1]研究了茜素红(ARS)与牛血清白蛋白(BSA)、人血清白蛋白(HSA)的结合反应。其吸附结合常数分别为:KBSA-ARS=3.950×104,KHSA-ARS=4.377×104。染料与蛋白的最大结合数分别为NARS∶NBSA=9∶1,NARS∶NHSA=7∶1。经光谱修正技术计算结合产物的实际摩尔吸光系数分别为εBSA-ARS(537nm)=2.517×104L.mol-1.cm-1,εHSA-ARS(519nm)=2.051×104L.mol-1.cm-1,检出限BSA为19mg/L,HSA为23mg/L。经探讨该结合反应机理符合Langmuir吸附聚集反应方程。     

同步荧光光谱法测定蛋白质

在模拟生理条件下,由于核苷类药物中间体氰基乙基尿嘧啶(CEU)与血清白蛋白相互作用,血清白蛋白的内源荧光发生特异性变化,且体系的同步荧光强度和溶液中血清白蛋白的浓度呈线性关系,据此提出以氰基乙基尿嘧啶为探针,用固定波长同步荧光光谱法测定人血清白蛋白(HSA)和牛血清白蛋白(BSA)的方法.在最佳试验条件下,体系的荧光强度与HSA和BSA的质量浓度分别在1.38～496.2 mg·L-1和1.56～624.0 mg·L-1范围内呈线性关系,检出限(3S/N)分别为0.045 mg·L-1和0.051 mg·L-1.方法应用于人血清及牛血清中HSA及BSA的测定,并以此样品为基体分别加入HSA及BSA标准溶液作回收试验,测得回收率在95.1%～102.5%之间,相对标准偏差(n=6)在0.43%～2.72%之间.     

β-环糊精微球对染料分子吸附作用研究

以环氧氯丙烷为交联剂,采取反相乳液聚合得到β-环糊精微球(EPI-β-CD),经丁二酸酐酰化后得到微球表面具有羧基的β-环糊精微球(SUC-EPI-β-CD),分别采用红外光谱、扫描电镜和激光粒度分析仪表征了其结构及形貌特征.选择染料亚甲基蓝(MB)和甲基橙(MO)作为模型分子,考察了所合成微球的吸附性能.结果表明:由于β-CD与两种染料分子的相互作用力不同,微球EPI-β-CD吸附亚甲基兰染料的能力强于甲基橙;两种微球对MB的吸附等温数据与Freundlich方程拟合较好,且均为优惠吸附;羧丁基酰化β-环糊精微球(SUC-EPI-β-CD)表现出较明显的pH敏感性.     

血清白蛋白的铅(Ⅱ)-抗坏血酸络合吸附波法测定

在pH9．2的H3BO3-NaOH缓冲溶液中,血清白蛋白使铅(Ⅱ)-抗坏血酸络合物在-0．51V(vs．SCE)处的络合吸附波电流降低,电流降低值与加入的牛血清白蛋白(BSA)或人血清白蛋白(HSA)的浓度在一定范围内呈线性关系。BSA在2～38μg／mL范围内,HSA在2～35μg／mL范围内呈线性关系,检出限均为1μg／mL。应用该法测定了人血清样品中蛋白质的含量,结果令人满意。     

N-乙烯吡咯烷酮/甲基丙烯酸-β-羟乙酯无规共聚物凝胶对蛋白质吸附机理的研究

用紫外分光光度法研究了牛血清白蛋白(BSA)在N-乙烯吡咯烷酮(NVP)/甲基丙烯酸-β-羟乙酯(HEMA)共聚物水凝胶上的吸附量.结果表明,BSA在凝胶上的吸附关系既可用Langmuir方程描述,又可用计量置换吸附Freundlich方程描述,且用Langmuir方程描述更为优越.研究了凝胶对BSA的吸附动力学行为,建立牛血清白蛋白的吸附动力学模型,发现用Bangharm方程对BSA吸附动力学曲线拟合程度较好,Bangharm方程可以较好地描述BSA在NVP/HEMA凝胶上的吸附动力学行为.     

芘与人血清白蛋白和牛血清白蛋白结合位点微环境极性的差异

利用芘(Pyr)的微环境极性探针性质, 采用稳态荧光光谱、 荧光共振能量转移技术结合分子对接法, 对比分析了Pyr分别与人血清白蛋白(HSA)和牛血清白蛋白(BSA)作用机制的差异. 结果表明, HSA和BSA中Pyr的I₁/I₃平均值分别为1.36和0.92; Pyr与HSA和BSA的结合常数分别为1.86×10⁷和1.71×10⁵ L/mol; Pyr与HSA和BSA中色氨酸残基表观距离分别为2.37和2.34 nm. Pyr在HSA和BSA中不同的结合位点位于ⅠB子域和ⅠA子域, 其结合位点周围氨基酸残基的极性是影响Pyr I₁/I₃值的主要原因之一. 实验证实Pyr与HSA和BSA结合作用位点处的微环境极性存在差异.     

活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用

以3.0 μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出 一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和 溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观 解离常数为5.26×10-5 mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶 电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。     

Preparation and chromatographic behavior of acetate-fiber filter rods with dye affinity ligands

Summary The dyes cibacron blue F3GA and reactive red 120 have been immobilized on acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorption of bovine serum albumin or human serum albumin on these matrixes was investigated and proved to conform to the Freundlich equation. In the static adsorption of human plasma the adsorption capacity for human serum albumin was 12.5 mg g⁻¹ fiber filter and one band was observed in SDS-PAGE electrophoresis of human serum albumin isolated by the immobilized cibacron blue F3GA filter rod. Ligand utilization efficiencies and breakthrough volumes were obtained for adsorption of HSA on the cibacron blue F3GA filter rod when the feed-rates were from 0.5 to 6 mL min⁻¹. In the affinity chromatography of human plasma the yield of human serum albumin was 1.27 g per 100 mL plasma.     

Removal of Endotoxin from Human Serum Albumin Solutions by Hydrophobic and Cationic Charged Membrane

Removal of end6toxin from medicine injection is very important, becauseendotoxin withpotential biological activity causes pyrogenic and shock reactions in' mammals-'on...intravenous injection even as law as "an6gram amounts. Endotoxin, a constituent ofpotential contaminant of physiological fluids and aqueous solutions 'and very stable atextreme temperature and PH values. For removing endotoxin from solutions ofbiomolecules, such as HSA, adsorption techniques are usedl.' Many methods forendot…     

Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate)

Continuous rod of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) was prepared by a free radical polymerization within the confines of a stainless-steel column. The epoxide groups of the rod were modified by a reaction with iminodiacetic acid (IDA) that affords the active site to form metal IDA chelates used for immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of bovine serum albumin on the Cu2+-IDA continuous rod column was investigated in the range from 5.0 to 9.0. Purification of lysozyme from egg white and human serum albumin (HSA) on the commercially available HSA solution were performed on the naked IDA and Cu2+-IDA continuous rod columns, respectively; and the purity of the obtained fractions was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.     

分析测试新成果(160~168)金属吸附剂用于去除血清蛋白及核苷检测

利用多糖与金属离子复合制备了一种高效的蛋白质吸附剂.海藻酸钠和羧甲基纤维素钠是两种富含羟基和羧基的多糖, 具有较强的金属亲和性.将其用钙离子交联后制备成金属-多糖复合材料, 进一步修饰铜离子, 得到蛋白质吸附剂.吸附剂对富含组氨酸的牛血红蛋白的吸附量可以达到33 g/g, 对少量组氨酸的牛血清白蛋白的吸附量也可以达到9.8 g/g.蛋白质吸附剂对人血血清进行两次吸附后, 可以去除其中98%的蛋白, 能够满足人血血清中核苷类物质的直接色谱进样检测.     

One-pot preparation of silica-supported hybrid immobilized metal affinity adsorbent with macroporous surface based on surface imprinting coating technique combined with polysaccharide incorporated sol--gel process

A simple and reliable one-pot approach using surface imprinting coating technique combined with polysaccharide incorporated sol-gel process was established to synthesize a new organic-inorganic hybrid matrix possessing macroporous surface and functional ligand. Using mesoporous silica gel being a support, immobilized metal affinity adsorbent with a macroporous shell/mesoporous core structure was obtained after metal ion loading. In the prepared matrix, covalently bonded coating and morphology manipulation on silica gel was achieved by using one-pot sol-gel process starting from an inorganic precursor, -glycidoxypropyltrimethoxysiloxane (GPTMS), and a functional biopolymer, chitosan (CS) at the atmosphere of imprinting polyethylene glycol (PEG). Self-hydrolysis of GPTMS, self-condensation, and co-condensation of silanol groups (Si-OH) from siloxane and silica gel surface, and in situ covalent cross-linking of CS created an orderly coating on silica gel surface. PEG extraction using hot ammonium hydroxide solution gave a chemically and mechanically stabilized pore structure and deactivated residual epoxy groups. The prepared matrix was characterized by using X-ray energy dispersion spectroscopy (EDX), scanning electron microscopy (SEM) and mercury intrusion porosimetry. The matrix possessed a high capacity for copper ion loading. Protein adsorption performance of the new immobilized metal affinity adsorbent was evaluated by batch adsorption and column chromatographic experiment using bovine serum albumin (BSA) as a simple model protein. Under the optimized coating conditions, the obtained macroporous surface resulted in a fast kinetics and high capability for protein adsorption, while the matrix non-charged with metal ions offered a low non-specific adsorption.     

染料壳聚糖微球的制备及其对人血清白蛋白吸附性能研究

人血清白蛋白(Human Serum Albumin,HSA)是血浆中含量最丰富的蛋白质,约占血浆总蛋白的60%.在人体内,HSA有许多重要的生理功能[1],临床上广泛应用于手术输血和危重病人补液,治疗创伤休克、烧伤、水肿和低白蛋白血症等,而且能增强人体抵抗能力,是迄今为止产量最大、临床用量最大     

Adsorption of Proteins with Tannin Modified Chitosan Magnetic Particles

Magnetic polymer particles have been widely used in biochemistry and medicine in recent years [1-4], mainly due to their property of relatively rapid and easy separation. There were many ways for preparation of magnetic particles [5-9]. We know natural polymer having convenient site such as-NH2,-COOH,-OH,-CONH2, etc. for the affinity ligand attachment. The literature reported chitosan as magnetic polymer matrix, dye as affinity ligand to purify bovine serum albumin and lysozyme[10l. Tannin, a natural product having multiple adjacent hydroxy groups, has extremely high affinity to adsorb protein or alkaloid. However, the information about tannin modified magnetic support is still sparse. Therefore, tannin modified chitosan magnetic particle was prepared and the adsorption of trypsin and aprotinin were studied.     

Protein adsorption equilibria and kinetics to a poly(vinyl alcohol)-based magnetic affinity support

A poly(vinyl alcohol)-based magnetic gel entrapping Fe3O4 colloids has been prepared by an emulsification-crosslinking method. The gel was modified with Cibacron blue 3GA, and thus a magnetic affinity support was produced. The adsorption equilibrium studies showed that the adsorption isotherm of lysozyme was nearly rectangular, with a capacity of 254 mg/ml, while the adsorption isotherm of bovine serum albumin obeyed the Henry's law. Uptake kinetics of the two proteins was investigated and analyzed with a pore diffusion model and a homogeneous diffusion model. Experimental results showed that the magnetic affinity gel had magnetic responsiveness and favorable properties in protein adsorption, and was mechanically and chemically stable.     

Protein C recognition by ion‐coordinated imprinted monolithic cryogels

The protein C imprinted monolithic cryogel was synthesized using 2‐hydroxyethyl methacrylate by redox cryo‐polymerization method. The prepared monolithic cryogel was characterized by Fourier transform infrared spectroscopy, swelling test, surface area measurements, and scanning electron microscopy. The nonimprinted cryogel was prepared as well for control. Adsorption of protein C from aqueous solutions was investigated in a continuous mode and several parameters affecting adsorption performance were optimized. The maximum protein C adsorption amount was 30.4 mg/g. The selectivity studies were performed by monolithic column studies and fast protein liquid chromatography, using hemoglobin and human serum albumin as competing proteins. The relative selectivity coefficients were 2.37 and 8.89 for hemoglobin and human serum albumin, respectively. Reusability was tested for ten consecutive adsorption–desorption cycles, and no significant change in adsorption capacity was recorded. A pseudo‐second‐order model was suitable to interpret kinetic data, and the Langmuir model suited the adsorption isotherms well.     

A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

1. INTRODUCTION Chitosan is a hydrolyzed derivative of chitin and belongs to a family of linear unbranched polysaccharides which contain large amounts of 1,4-linked-2-amino-2-deoxy-β-D-glucan residues. The presence of free amine groups in chitosan enhances the solubility and reactivity of this polymer. Interest in modifying chitosan by using glutaraldehyde has recently increased. The derivatized polymers have been employed for many applications [1~2], including protein immobilization…