双组分蛋白质离子交换吸附行为研究
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摘要
离子交换色谱是目前应用最广泛的液相色谱技术,为了更好地指导离子交换色谱技术在蛋白质大分子分离纯化中的实际应用,本文选取牛血清白蛋白(BSA)和牛血红蛋白(Hb)作为双组分模型蛋白,Q Sepharose FF作为离子交换介质,对其吸附行为进行了一系列的研究,主要包括以下几个方面:
1.通过不同pH值、离子强度、缓冲液体系和不同蛋白浓度比条件下的热力学平衡研究,证明了双组分吸附中的竞争行为的存在,BSA在吸附中占主导地位。BSA的吸附主要受液相条件影响,而Hb的吸附主要由BSA的竞争吸附决定。双组分蛋白离子交换次序吸附的研究证明吸附顺序对蛋白质的竞争吸附有一定影响。
2.用单组分空间质量作用模型(SMA模型)参数对不同条件下双组分蛋白离子交换吸附等温线进行拟合,其结果表明多组分SMA模型能够用于双组分蛋白竞争吸附平衡的描述,且对强吸附蛋白组分的拟合效果较好。对同一蛋白来说,多组分SMA模型在有利于其竞争吸附条件下的拟合精度提高,反之精度降低。
3.单双组分动力学研究结果表明BSA的传质扩散速率大于Hb。BSA在双组分条件下的传质扩散速率与单组分条件下相同,而Hb在双组分中的传质则进一步被BSA抑制,从而BSA先与吸附位点作用,Hb则只能与没被BSA占据的位点作用。
4.通过色谱穿透实验实现了不同的双组分蛋白浓度比的条件下蛋白混合溶液的高效分离,收集到的有高纯度,高回收率的单组分Hb蛋白。本文通过对双组分蛋白质离子交换吸附过程进行系统的研究,深入了解了双组分蛋白质在离子交换吸附过程中的竞争关系及其影响因素,并证实双组分蛋白质离子交换吸附热力学平衡研究和动力学研究对其实际离子交换色谱分离行为和效果具有很好的指导作用。
Ion-exchange chromatography is the most widely used liquid chromatographic technique. In order to provide a practical guidance to the development of chromatographic process for the purification of protein, binary adsorption of proteins on anion-exchanger, Q Sepharose FF, was investigated in the well-mixed container and chromatographic column using bovine serum albumin (BSA) and bovine hemoglobin (Hb) as model proteins. The following research works were carried out:
1. Proteins adsorption equilibria were conducted at different buffer pHs, ionic strengths, buffer constituents and amount ratios of proteins, respectively. It was confirmed that the competitive adsorption occurred in the binary adsorption of BSA and Hb on Q Sepharose FF. In this pair of proteins, adsorption of BSA was dominant and was mainly influenced by buffer while adsorption of Hb was mainly determined by BSA. Experimental evidence of sequential adsorption of binary proteins indicated that competitive adsorption behavior of BSA and Hb was influenced by order of adsorption.
2. Parameters of steric mass-action (SMA) model obtained from the adsorption equilibria of single-component protein were used to fit the isotherms of binary-component adsorption to ion-exchange media at different conditions. The result indicated that SMA model could be used to describe binary adsorption equilibria, and a better fitting curve could be obtained for stronger adsorbed BSA. For a single protein component, better fitting curves could be obtained in conditions in favor of its competitive adsorption.
3. Dynamic results of both single and binary components showed BSA had a higher diffusivity inside gel pores than Hb. Furthermore, BSA exhibited similar intraparticle diffusivity in the adsorption of both single and binary proteins, while the intraparticle diffusive rate of Hb was further restrained by BSA. In the binary adsorption of proteins, BSA bound favorably to adsorption sites on anion exchanger, and Hb only interacted with the adsorption sites left by BSA.
4. Breakthrough experiments on chromatography realized high performance separation of binary component proteins (BSA and Hb) at different protein concentration ratios. Hb solution was collected with high purity and high recovery.
The competition of binary adsorption and its influence facts were better understood by investigating of binary adsorption of model proteins adsorption to ion-exchange media at different conditions with comprehensive researches. Researches on adsorption equilibrium and adsorption dynamics of binary adsorption can guide the separation of ion-exchange chromatography well.
1.通过不同pH值、离子强度、缓冲液体系和不同蛋白浓度比条件下的热力学平衡研究,证明了双组分吸附中的竞争行为的存在,BSA在吸附中占主导地位。BSA的吸附主要受液相条件影响,而Hb的吸附主要由BSA的竞争吸附决定。双组分蛋白离子交换次序吸附的研究证明吸附顺序对蛋白质的竞争吸附有一定影响。
2.用单组分空间质量作用模型(SMA模型)参数对不同条件下双组分蛋白离子交换吸附等温线进行拟合,其结果表明多组分SMA模型能够用于双组分蛋白竞争吸附平衡的描述,且对强吸附蛋白组分的拟合效果较好。对同一蛋白来说,多组分SMA模型在有利于其竞争吸附条件下的拟合精度提高,反之精度降低。
3.单双组分动力学研究结果表明BSA的传质扩散速率大于Hb。BSA在双组分条件下的传质扩散速率与单组分条件下相同,而Hb在双组分中的传质则进一步被BSA抑制,从而BSA先与吸附位点作用,Hb则只能与没被BSA占据的位点作用。
4.通过色谱穿透实验实现了不同的双组分蛋白浓度比的条件下蛋白混合溶液的高效分离,收集到的有高纯度,高回收率的单组分Hb蛋白。本文通过对双组分蛋白质离子交换吸附过程进行系统的研究,深入了解了双组分蛋白质在离子交换吸附过程中的竞争关系及其影响因素,并证实双组分蛋白质离子交换吸附热力学平衡研究和动力学研究对其实际离子交换色谱分离行为和效果具有很好的指导作用。
Ion-exchange chromatography is the most widely used liquid chromatographic technique. In order to provide a practical guidance to the development of chromatographic process for the purification of protein, binary adsorption of proteins on anion-exchanger, Q Sepharose FF, was investigated in the well-mixed container and chromatographic column using bovine serum albumin (BSA) and bovine hemoglobin (Hb) as model proteins. The following research works were carried out:
1. Proteins adsorption equilibria were conducted at different buffer pHs, ionic strengths, buffer constituents and amount ratios of proteins, respectively. It was confirmed that the competitive adsorption occurred in the binary adsorption of BSA and Hb on Q Sepharose FF. In this pair of proteins, adsorption of BSA was dominant and was mainly influenced by buffer while adsorption of Hb was mainly determined by BSA. Experimental evidence of sequential adsorption of binary proteins indicated that competitive adsorption behavior of BSA and Hb was influenced by order of adsorption.
2. Parameters of steric mass-action (SMA) model obtained from the adsorption equilibria of single-component protein were used to fit the isotherms of binary-component adsorption to ion-exchange media at different conditions. The result indicated that SMA model could be used to describe binary adsorption equilibria, and a better fitting curve could be obtained for stronger adsorbed BSA. For a single protein component, better fitting curves could be obtained in conditions in favor of its competitive adsorption.
3. Dynamic results of both single and binary components showed BSA had a higher diffusivity inside gel pores than Hb. Furthermore, BSA exhibited similar intraparticle diffusivity in the adsorption of both single and binary proteins, while the intraparticle diffusive rate of Hb was further restrained by BSA. In the binary adsorption of proteins, BSA bound favorably to adsorption sites on anion exchanger, and Hb only interacted with the adsorption sites left by BSA.
4. Breakthrough experiments on chromatography realized high performance separation of binary component proteins (BSA and Hb) at different protein concentration ratios. Hb solution was collected with high purity and high recovery.
The competition of binary adsorption and its influence facts were better understood by investigating of binary adsorption of model proteins adsorption to ion-exchange media at different conditions with comprehensive researches. Researches on adsorption equilibrium and adsorption dynamics of binary adsorption can guide the separation of ion-exchange chromatography well.
引文
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[2]俞俊棠,唐孝宣,生物工艺学(上册),上海:华东化工学院出版社,1991, 242~251
[3]刘国诠,生物工程下游技术,北京:化学工业出版社,1993, 141~142
[4]师治贤,王俊德,生物大分子的液相色谱分离和制备,北京:科学出版社,1999, 2~8
[5]Himmelhoch S R, Chromatography of proteins on ion-exchange adsorbents, Methods in Enzymology, 1971, 22: 273~286
[6]秦启宗,毛家骏,金忠浩,化学分离法,北京:原子能出版社,1984, 126~138
[7]Carta G, Ubiera A R, Past TM, Protein mass transfer kinetics in ion exchange media:measurements and interpretations, Chemical Engineering and Technology, 2005, 28(11): 1252~1264
[8]孙彦,生物分离工程(第二版),北京:化学工业出版社,2005, 271~279
[9]Hunter A K, Carta G, Protein adsorption on novel acrylamido-based polymeric ion-exchangers: IV. Effects of protein size on adsorption capacity and rate, Journal of Chromatography A, 2002, 971(1-2): 105~116
[10]Yuan Y, Oberholzer M R, Lenhoff A M, Size does matter: electrostatically determined surface coverage trends in protein and colloid adsorption, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2000, 165(1-3): 125~141
[11]Conder J R, Hayek B O, Adsorption and desorption kinetics of bovine serum albumin in ion exchange and hydrophobic interaction chromatography on silica matrices, Biochemical Engineering Journal, 2000, 6(3): 225~232
[12]Katiyar A, Ji L, Smirniotis P, et al., Protein adsorption on the mesoporous molecular sieve silicate SBA-15: effects of pH and pore size, Journal of Chromatography A, 2005, 1069(1): 119~126
[13]Bosma J C, Wesselingh J A, pH dependence of ion-exchange equilibrium of proteins, AIChE Journal, 1998, 44(11): 2399~2409
[14]Peng Z G, K Hidajat K, Uddin M S, Adsorption of bovine serum albumin on nanosized magnetic particles, Journal of Colloid and Interface Science, 2004, 271(2): 277~283
[15]Zhang S, Sun Y, Ionic strength dependence of protein adsorption to dye-ligand adsorbents, AIChE Journal, 2002, 48(1): 178~186
[16]Janzen R, Unger K K, Müller W, et al., Adsorption of proteins on porous and non-porous poly(ethyleneimine) and tentacle-type anion exchangers, Journal of Chromatography A, 1990, 522: 77~93
[17]Langmuir I, The adsorption of gases on plane surface of glass, mica and platinum,Journal of the American Chemical Society, 1918, 40: 1361~1403
[18]周盈,蛋白质离子交换吸附平衡理论研究:[硕士学位论文],天津;天津大学,2003
[19]Aboudzadeh M R, Aboudzadeh N, Zhu J W, et al., Binary protein adsorption to DEAE sepharose FF, Korean Journal of Chemical Engineering, 2007, 24(4): 641~647
[20]Cano T, Offringa N, Willson R C, The effectiveness of three multi-component binding models in describing the binary competitive equilibrium adsorption of two cytochrome b(5) mutants, Journal of Chromatography A, 2007, 1144(2): 197~202
[21]Levan M D, Vermeulen T, Binary Langmuir and Freundlich isotherms for ideal adsorbed solutions, Journal of Physical Chemistry, 1981, 85: 3247~3250
[22]Li Y, Pinto N G, Model for ion-exchange equilibria of macromolecules in preparative chromatography, Journal of Chromatography A, 1995, 702: 113~123
[23]Brooks C A, Cramer S M, Steric mass-action ion exchange: Displacement profiles and induced salt gradients, AIChE Journal, 1992, 38(12): 1969~1978
[24]Velayudhan A, Studies in Non-linear Chromatography, Doctoral Disseratation, Yale University, New Haven, CT, 1990
[25]Velayudhan A, Horvath C, Preparative chromatography of proteins: Analysis of the multivariant ion-exchange formalism, Journal of Chromatography A, 1988, 443: 13~29
[26]苏雪丽,蛋白质静电吸附平衡与动力学的研究:[博士学位论文],天津;天津大学,2005
[27]Hill T L, An introduction to Statistical Thermodynamics, Addison-Wesley Pub Co, Reading, Mass, 1960, 382~388
[28]周笑鹏,蛋白质吸附平衡和动力学理论研究:[博士学位论文],天津;天津大学,2006
[29]Boardman N K, Partridge S M, Separation of neutral proteins on ion-exchange resins, Biochemical Journal, 1955, 59: 543~552
[30]Cysewski P, Jaulmes A, Lemque R, et al., Multivalent ion-exchange model of biopolymer chromatography for mass overloaded conditions, Journal of Chromatography A, 1991, 548: 61~79
[31]Bellot J C, Condoret J S, Theoretical study of the ion-exchange preparative chromatography of a two-protein mixture, Journal of Chromatography A, 1993, 635: 1~17
[32]Bellot J C, Condoret J S, Modelling of liquid chromatography equilibria, Process Biochemistry, 1993, 28: 365~376
[33]Kopaciewicz W, Rounds M A, Fausnaugh J, et al., Retention model for high-performance ion-exchange chromatography, Journal of Chromatography A, 1983, 266: 3~21
[34]Rounds M A, Regnier F E, Evaluation of a retention model for high-performance ion-exchange chromatography using two different displacing salts, Journal of Chromatography A, 1984, 283: 37~45
[35]Roth C M, Unger K K, Lenhoff A M, Mechanistic model of retention in proteinion-exchange chromatograph, Journal of Chromatography A, 1996, 726 (1-2): 45~56
[36]Bosma J C, Wesselingh J A, Available area isotherm, AIChE Journal, 2004, 50(4): 848~853
[37]Staahlberg J, Electrostatic retention model for ion-exchange chromatography, Analytical Chemistry, 1994, 66(4): 440~449
[38]Li Y L, Pinto N G, Influence of lateral interactions on preparative protein chromatography, Journal of Chromatography A, 1994, 658: 445~457
[39]Staahlberg J, Joensson B, Horvath C, Theory for electrostatic interaction chromatography of proteins, Analytical Chemistry, 1991, 63(17): 1867~1874
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