蛋白质水力学半径的测定及用于蛋白质变性过程的实时监控

采用纳米粒度分析仪Zetasizer Nano测定了牛血清白蛋白（BSA）的水力学半径,考察了pH、离子强度、表面活性剂等的影响,并用于BSA热变性和脲变性过程的实时跟踪。随pH增大,BSA水力学半径呈“U”形变化趋势;酸性条件下,分子膨胀,随着盐浓度升高,BSA分子先小幅减小后显著增大;中性pH范围,离子强度对蛋白质分子尺寸影响很小,蛋白质性质相对稳定。离子型表面活性剂在蛋白质表面吸附,从而影响BSA水力学半径。通过水力学半径的测量实时跟踪蛋白质变性过程的分子变化,发现增加热变性中离子强度可加快变性速率,SDS加入增大了BSA变性温度Tm。脲在促使BSA分子扩张的同时,对链伸展有抑制作用;在DTT存在下,BSA的水力学半径随着时间变化逐渐增大。结果表明,简便的水力学半径测量可以用于蛋白质大小的表征,并可实时跟踪蛋白变性过程的分子尺度变化。     

反胶团萃取蛋白质特性的研究

实验将一种阴离子表面活性剂（AOT）和一种阳离子表面活性剂（Aliquat336）分别溶于异辛烷（isooctane）中，构成了两种不同的反胶团体系．通过用两种不同的反胶团溶液萃取六种蛋白质的实验，研究了水相pH值及离子强度对反胶团体系中水含量Wo和蛋白质萃取率影响的规律．实验发现不论哪种反胶团体系，水相pH值对Wo的影响都不大，但pH值却对蛋白质萃取率有很大的影响，对AOT反胶团体系而言，随着pH值的降低，蛋白质的萃取率升高；对Aliquat336反胶团体系而言，随着水相pH值的降低，蛋白质的萃取率下降．随着离子强度的增大，AOT反胶团体系的水含量大幅度地降低；而对Aliquat336体系水含量的影响很小，但水相离子强度对蛋白质萃取率的影响是相同的，随着离子强度的增大，两种体系对蛋白质的萃取率均下降。     

蛋白质zeta电位与离子交换层析容量因子的相关性研究

针对蛋白质离子交换层析分离过程预测的困难,选择zeta电位作为中间参数,建立zeta电位与层析容量因子之间的关联.选择牛血清白蛋白(BSA)作为模型蛋白质,考察了pH、离子浓度、离子种类等对蛋白质zeta电位的影响,证实了zeta电位作为蛋白质表面电荷特性表征参数的可行性.以阴离子交换Sepharose FF DEAE为模型层析介质,测定了BSA在不同pH和盐浓度下的容量因子.综合考虑蛋白质和介质两方面因素,选择两者zeta电位乘积(-ζ1×ζ2)作为中间参数,对于不同pH和盐浓度的流动相,(-ζ1×ζ2)与容量因子的对数呈现良好的线性关系,实现了由简便的zeta电位分析预估宏观离子交换层析分离行为的目的,该关联关系的建立将有助于层析分离过程的优化设计.     

裙带菜吸附水溶液中镍离子的特性及吸附平衡模型

本文实验研究了裙带菜对废水中重金属镍离子的吸附特性和吸附平衡模型。实验表明裙带菜对镍离子的吸附能力最高达O．76mmol/g(干裙带菜)。在恒定pH条件下,Langmuir方程可用来描述吸附容量与平衡浓度的关系。但吸附容量受溶液pH值影响很大,在pH2．O～6．O范围内,pH值越低吸附量越小,pH在5.5～6.O时有较大吸附容量。竞争吸附模型可以很好地描述溶液中pH值和离子浓度对吸附的影响,可以预测再生条件。     

氨基表面修饰的高分子乳液微球对牛血白质BSA吸附性能的研究

以苯乙烯St和甲基丙烯酸环氧丙酯GMA为单体,两性偶氮类化合物V50为引发剂,运用功能单体多段加料的无皂乳液聚合法制备亲和高分子乳液基球PSG,并选择双氨基封端的聚乙二醇作为空间臂分子,进行表面修饰反应,考察了高分子乳液微球对牛血蛋白BSA的吸附性能,并着重研究了吸附时间和温度、BSA初始浓度、离子强度等因素对吸附反应的影响。实验结果表明,空间臂表面修饰的高分子微球作为载体材料,固定药物分子等生物活性物质,可通过控制吸附条件,如pH,温度,反应时间等,减少蛋白质分子的非特异性吸附,且保持生化分子的较高活性。     

氨基酸中空纤维膜分配色谱模型

以色氨酸为例,考虑氨基酸解离和在中空纤维膜内扩散传质建立了氨基酸中空纤维膜分配色谱理论模型.该模型采用完全独立的模型参数,模拟结果与实验数据吻合较好,验证了模型的可靠性.理论计算表明流动相的pH值与局部氨基酸浓度密切相关;理论和实验结果表明,流动相离子强度对氨基酸的保留行为有很大影响,采用梯度洗脱的方式可以改善洗脱峰形,提高色谱柱的分离度.     

酰胺功能化SBA-15对铀(Ⅵ)的吸附研究

本研究将1-[3-(三甲氧基硅烷)丙基]脲基团嫁接在介孔材料SBA-15上,在15℃下实验研究了pH值、离子强度、时间对该材料对铀吸附的影响。介孔材料经过XRD,N2吸附脱附进行表征。结果表明,该材料对UO22+的吸附受pH值、离子强度影响较大,材料对铀酰离子吸附模型符合Langmuir模型,吸附容量达78 mg/mL。     

基于动态光散射的溶菌酶水力学直径研究

采用Zeta电位和粒度分析仪测量了溶菌酶的水力学直径,研究了pH值、离子强度和脲浓度对其大小的影响.随着 pH值增加,溶菌酶的水力学直径呈” W”形分布;在极端的碱性条件下,溶菌酶水力学直径随离子强度的增加而显著变大;中性pH值时,水力学直径随离子强度的增加变化不大.脲对溶菌酶具有双重作用,随着溶液中脲浓度的增加,溶菌酶的水力学直径先减小后增大.结果表明,动态光散射技术可以很好地应用于研究蛋白质分子的均一性和稳定性,同时也可以通过水力学直径来表征pH值、离子强度和脲对溶菌酶的影响.     

黄铜矿物表面吸附细菌的研究

对黄铜矿表面吸附细菌的特性进行了研究。结果表明,氧化亚铁硫杆菌在黄铜矿表面的的吸附平衡符合Freundlich方程。在研究范围内,菌龄为对数生长后期、pH值1.5～3.0、离子强度50～100mg/mL和非离子表面活性剂质量分数在0.005%～0.1%时更有利于细菌在矿物表面的吸附,但温度对吸附影响不大。采用拟二阶反应模型考察了吸附动力学,并计算了此动力学模型的速率常数。进一步研究表明,二级反应模型和实验数据之间有较好的相关性。     

阳离子反胶团体系萃取牛血清白蛋白

采用一种新的阳离子反胶团体系(CTAB/异辛烷-正戊醇),研究了反胶团萃取牛血清白蛋白(BSA).用单变量法考察了表面活性剂浓度、水相pH值、离子种类和浓度、萃取时搅拌速率和油水比对BSA萃取率的影响.实验表明,静电作用力是该萃取过程的主要动力,随水相pH值增大,BSA的萃取率逐渐升高;对于不同种类的离子,基本随其浓度增大,萃取率下降.在优化的操作条件下,BSA萃取率可达98%以上,因此,CTAB/异辛烷-正戊醇反胶团体系适合BSA的萃取.     

Analysis of steric mass-action model for protein adsorption equilibrium onto porous anion-exchange adsorbent

The ion-exchange equilibrium of bovine serum albumin (BSA) to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments at pH values ranging from 5.26 to 7.6 and ionic strengths from 10 to 117.1 mmol/l. Using the unadjustable adsorption equilibrium parameters obtained from batch experiments, the applicability of the steric mass-action (SMA) model was analyzed for describing protein ion-exchange equilibrium in different buffer systems. The parametric sensitivity analysis was performed by perturbing each of the model parameters, while holding the rest constant. The simulation results showed that, at high salt concentrations or low pHs close to the isoelectric point of the protein, the precision of the model prediction decreased. Parametric sensitivity analysis showed that the characteristic charge and protein steric factor had the largest effects on ion-exchange equilibrium, while the effect of equilibrium constant was about 70%-95% smaller than those of characteristic charge and steric factor under all conditions investigated. The SMA model with the relationship between the adjusted characteristic charge and the salt concentration can well predict the protein adsorption isotherms in a wide pH range from 5.84 to 7.6. It is considered that the SMA model could be further improved by taking into account the effect of salt concentration on the intermolecular interactions of proteins.     

蛋白质离子交换的空间质量作用模型分析

引　言离子交换层析具有分离容量大、选择性高和介质种类多、价格适中等特点 ,是蛋白质类生物大分子分离纯化的主要手段[1] .离子交换或吸附平衡研究是离子交换层析过程设计和优化分析的基础 ,也是分离实践中评价和选择离子交换介质的重要步骤 .目前已提出多种模型用于描述离子交换平衡 ,其中应用最多的是基于Ｌａｎｇｍｕｉｒ理论的经验模型[2 ] .但是 ,Ｌａｎｇｍｕｉｒ模型不能直接反映溶液离子强度对平衡的影响 ,不利于在盐浓度梯度洗脱的离子交换层析过程中的应用 .近年来 ,基于离子交换反应和蛋白质空间屏蔽作用的空间质量作用模型受…     

空间质量作用模型预测小分子离子交换平衡

The steric mass-action (SMA) model has been widely reported in the literature for ion-exchange and metal-affinity interaction adsorption equilibrium of biomacromolecules. In this paper, the usefulness of SMA model is analyzed for describing micromolecule ion-exchange equilibrium onto cation exchangers, CM Sephadex C-25 and Streamline SP. Batch adsorption experiments with ephedrine hydrochloride as a model adsorbate are carried out to determine the model parameters, that is, steric factor, characteristic charge and equilibrium constant. The result shows that the SMA model parameters of micromolecule cannot be obtained using the nonlinear least-square fitting method as protein‘s due to the remarkable difference between the molecular mass and dimension of micromolecule and protein. It is considered that the small size of the adsorbates dealt with in this study justifies the neglect of steric hindrances arising from adsorbate bulkiness. Thus, the three-parameter SMA model is reduced to two-parameter one (i.e., steric factor is equal to zero) for describing micromolecule ion-exchange equilibrium. It is found that the equilibrium constant for CM Sephadex C-25 increases with increasing ionic strength, while the equilibrium constant for Streamline SP shows an opposite trend. This is probably due to the remarkable difference between the physicalpro perties of the two adsorbents. Then, the relationship between the equilibrium constant and ionic strength is described by an expression. The computer simulations show that, the theoretical model with the correlation is promising in the prediction of micromolecule adsorption decrease with increasing ionic strength in a wide range of salt concentration.     

吸附密度对蛋白质在离子交换吸附剂中孔扩散系数的影响

通过间歇吸附动力学实验,采用孔扩散模型研究了牛血清白蛋白和γ-球蛋白在阴离子交换剂中的扩散行为,考察了蛋白质初浓度和平均吸附密度对孔扩散系数的影响.结果表明,随蛋白质初浓度和平均吸附密度增大,孔扩散系数均呈指数下降,且蛋白质分子尺寸越大下降趋势越明显.     

阴离子交换剂DEAE-Spherodex M的蛋白质吸附性能

通过间歇吸附实验,采用Langmuir等温方程和改进的平行扩散模型,研究了牛血清白蛋白和γ 球蛋白在阴离子交换剂DEAE SpherodexM中的静态和动态吸附行为,分析了吸附过程的热力学参数。研究发现,阴离子交换剂的吸附平衡是吸热反应,对于较宽分子尺寸范围的蛋白质,DEAE SpherodexM均具有较高的离子交换吸附速率。研究表明,DEAE SpherodexM具有较好的机械强度、化学和热稳定性、稳定的功能基、较高的交换容量和较快的离子交换速度,是一种较好的蛋白质分离介质。     

Packed-microtubes model for flowthrough chromatography of protein

A mathematical model for flowthrough (perfusion) chromatography, namely packed-microtubes (PMT) model, has been proposed for a column packed with biporous (BiP) anion exchanger in which the mesopores and flowthrough pores are created with liquid and solid porogens, respectively. The model is established based on the assumption that the BiP particle is made up of packed microtubes. Bovine serum albumin (BSA) is used as a model protein and three kinds of anion exchangers (i.e., mesoporous, macroporous and BiP resins) are used as adsorbents to determine the model parameters and to evaluate the model. Adsorption equilibrium and finite bath experiments are performed to determine the adsorption isotherms and kinetics parameters. Both the bound amounts of the protein on the surface of the mesopores and macropores are experimentally determined and taken into account in the mathematical model. With all the model parameters determined by independent experiments or calculated from available correlations, model simulations are performed and compared with the experimentally determined breakthrough profiles of the BiP column. It is found that the model predictions agree reasonably well with the experimental data obtained under various conditions and the PMT model fit experimental data better than the modified double linear driving force (LLDF) model proposed by Leitão and Rodriogues (1999. Biochemical Engineering Journal 3, 131) in which the adsorbent particle is considered to be made up of packed microparticles. The results indicate that the PMT model is more reasonable for this kind of BiP adsorbent.     

离子强度和溶质浓度对蛋白质在Q Sepharose FF中吸附动力学的影响

采用孔扩散模型, 模拟不同盐浓度和不同蛋白质初始浓度条件下,吸附牛血清白蛋白(BSA)的动态吸附曲线并获得孔扩散系数;考察了离子强度和溶质浓度对蛋白质在阴离子交换剂Q Sepharose FF中吸附动力学的影响.结果表明,蛋白质的孔扩散系数随初始浓度的增大而下降;在氯化钠浓度小于0.10 mol•L^-1的范围内,蛋白质的孔扩散系数随着盐浓度的增加而增大,但当盐浓度增大到0.15 mol•L^-1时又有所降低,表明存在着一个最佳的离子强度,使蛋白质的孔扩散系数最大.     

Modeling of protein adsorption to DEAE sepharose FF: Comparison of data with model simulation

The equilibrium and kinetic characteristics of the adsorption of human serum albumin (HSA) and ovalbumin (OVA) to the DEAE Sepharose FF weak anion exchanger were experimentally determined. The rate for protein adsorption was simulated with two different models, the first being based on a single lumped kinetic parameter, while the second includes the individual mass transfer processes occurring prior to the adsorption intervention, i.e., diffusion across the liquid film surrounding individual particles and diffusion within the ion exchanger particle itself. The actual adsorption of OVA to DEAE Sepharose FF in fully mixed stirred vessels and in packed bed columns was consistent with both models. In the case of HSA, however, the adsorption profile in an agitated vessel was consistent only with the pore diffusion model and neither model could correctly predict the latter part of the breakthrough profile observed in packed bed experiments.     

A modified Langmuir model for the prediction of the effects of ionic strength on the equilibrium characteristics of protein adsorption onto ion exchange/affinity adsorbents

An empirical model modified from the Langmuir isotherm model to account for the effects of ionic strength on the equilibrium characteristics of protein adsorption onto ion exchange/affinity adsorbents has been proposed and tested against experimental and literature data. The equilibrium isotherms for BSA adsorption onto a polystyrenic anion exchanger, Diaion HPA25, were established for five different NaCl concentrations at 25°C, pH 7.0. The apparent Langmuir parameters in the new model (q_m′ and K_d′), which replace the Langmuir parameters (q_m and K_d) in the original Langmuir model, were determined by non-linear curve fitting. The proposed model has been shown to be applicable to various protein/adsorbent systems.     

Adsorption of BSA on DEAE-Dextran: Equilibria

Abstract

Equilibrium isotherms for adsorption of BSA on DEAE-dextran have been determined experimentally. They were little affected by the initial concentration of BSA but were considerably affected by pH. They were correlated by the Langmuir equation when pH ≥ 5.05 and by the Freundlich equation at pH 4.8 (≈ pI). The maximum amounts of inorganic anion exchanged for BSA were 1 and 0.4% of the exchange capacity of the ion exchanger at pH 6.9 and 4.8, respectively. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm but was shifted to the right. When the concentration of NaCl was 200 mol/m³, BSA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO₄ ^2? -form resin was about 3.5 times larger than that with buffer (pH 6.9). The amount of BSA adsorbed on univalent-form resin decreased when the liquid-phase equilibrium concentration of BSA was increased.