蛋白质吸附平衡和动力学理论研究
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摘要
蛋白质的离子交换平衡和动力学行为在生物分离过程的设计和优化中起着十分重要的作用。然而,离子交换色谱理论尚不完善,围绕蛋白质离子交换色谱理论,本文展开了以下的研究工作:
1.采用统计热力学模型(ST模型)研究了蛋白质(BSA和Hb)在阴离子交换剂DEAE Spherodex M上的吸附平衡行为。结果表明,ST模型可以很好地描述不同离子强度条件下单组分和双组分蛋白质的吸附平衡行为。随着离子强度增高,吸附相伴离子对蛋白质与吸附剂之间和蛋白质分子间静电力的屏蔽作用增大。在相同的离子强度条件下,BSA与DEAE Spherodex M之间的静电引力较强;BSA分子之间的静电斥力大于Hb分子之间的静电斥力。在相同的离子强度条件下的Hb-BSA双组分吸附体系中,BSA与吸附界面静电吸引作用强,吸附竞争力强。由ST模型参数1/αi和k ii分别计算得到蛋白质的有效电荷数基本一致。
2.采用ST模型进而研究了BSA和Hb在阳离子交换剂SP Sepharose FF的吸附平衡行为。研究结果表明,随着离子强度增高,吸附相伴离子对蛋白质与吸附剂之间和蛋白质分子间静电力的屏蔽作用增大;而溶液pH值的升高引起蛋白质与吸附界面间的静电引力及蛋白质分子间静电斥力减弱。在相同的离子强度和pH值条件下,Hb与SP Sepharose FF之间的静电引力较强;Hb分子之间的静电斥力大于BSA分子之间的静电斥力。此时,在Hb-BSA双组分吸附体系中,Hb与吸附界面静电吸引作用强,吸附竞争力强。
3.建立了考虑吸附剂的粒径分布(PSD)的有效孔扩散模型(EPDM)、表面扩散模型(SDM)、Maxwell-Stefan模型(MSM)。通过蛋白质的间歇吸附动力学实验,研究了蛋白质(BSA)在阳离子交换剂(SP Sepharose FF)孔内的扩散。结果表明,这三种模型均能很好地拟合蛋白质的动态吸附曲线;但由于传质驱动力不同,各模型对孔内蛋白质沿吸附剂径向浓度分布图的预测存在较大差异。
4.利用共聚焦激光扫描显微镜(CLSM)直接观测了蛋白质在吸附剂颗粒内部的径向浓度分布情况。通过观测结果与考虑吸附剂PSD的模型(EPDM, SDM和MSM)拟合吸附剂孔内蛋白质径向浓度分布结果的比较,可以看出在目前的研究体系中SDM能够比EPDM更好地预测孔内蛋白质浓度分布图,MSM模型对孔内蛋白质浓度分布的预测结果介于另外两种模型之间。
5.由于在使用共聚焦显微镜观测吸附剂孔内蛋白质时,需要用荧光色素标
Ion-exchange equilibria and intraparticle mass transfer of protein in chromatography are of great importance for the design and optimization of bioseparation processes. Compared with the extensive application of ion-exchange chromatography in biotechnology, the mechanism and process analysis of ion-exchange system are still in the primary state. The details in this work are summarized as follows.
1. The adsorption equilibria of bovine serum albumin (BSA) and hemoglobin (Hb) to anion exchanger, DEAE Spherodex M, were studied by using statistical thermodynamic model (ST model). The ST model can well describe single and two-component protein equilibrium on the anion exchanger at different ionic strengths. The shielding effect of the co-ions in adsorption phase on the electrostatic interaction among protein molecules and adsorbents increases with an increase of ionic strength. At the same ionic strength, the electrostatic attraction between BSA molecules and DEAE Spherodex M is stronger, and the electrostatic repulsion among BSA molecules is stronger than that among Hb molecules. In two-component adsorption system, the electrostatic attraction between BSA molecules and adsorbent is stronger, BSA is more competitive. The effective protein charge values calculated respectively from ST model parameters 1/αiand k ii are consistent.
2. The adsorption equilibria of BSA and Hb to cation exchanger, SP Sepharose FF, were also studied by using ST model. The shielding effect of the co-ions in adsorption phase on the electrostatic interaction among protein molecules and adsorbents increases with increasing ionic strength. Moreover, the electrostatic interaction among protein molecules and adsorbents decreases with increasing pH. At the same pH values and ionic strengths, the electrostatic attraction between Hb molecules and SP Sepharose FF is stronger, and the electrostatic repulsion among Hb molecules is stronger than that among BSA molecules. In two-component adsorption system, the electrostatic attraction between Hb molecules and adsorbents is stronger, so Hb is more competitive.
3. The ion-exchange adsorption kinetics of BSA to the cation exchanger, SP Sepharose FF, was studied by batch adsorption experiments. Three diffusion models
1.采用统计热力学模型(ST模型)研究了蛋白质(BSA和Hb)在阴离子交换剂DEAE Spherodex M上的吸附平衡行为。结果表明,ST模型可以很好地描述不同离子强度条件下单组分和双组分蛋白质的吸附平衡行为。随着离子强度增高,吸附相伴离子对蛋白质与吸附剂之间和蛋白质分子间静电力的屏蔽作用增大。在相同的离子强度条件下,BSA与DEAE Spherodex M之间的静电引力较强;BSA分子之间的静电斥力大于Hb分子之间的静电斥力。在相同的离子强度条件下的Hb-BSA双组分吸附体系中,BSA与吸附界面静电吸引作用强,吸附竞争力强。由ST模型参数1/αi和k ii分别计算得到蛋白质的有效电荷数基本一致。
2.采用ST模型进而研究了BSA和Hb在阳离子交换剂SP Sepharose FF的吸附平衡行为。研究结果表明,随着离子强度增高,吸附相伴离子对蛋白质与吸附剂之间和蛋白质分子间静电力的屏蔽作用增大;而溶液pH值的升高引起蛋白质与吸附界面间的静电引力及蛋白质分子间静电斥力减弱。在相同的离子强度和pH值条件下,Hb与SP Sepharose FF之间的静电引力较强;Hb分子之间的静电斥力大于BSA分子之间的静电斥力。此时,在Hb-BSA双组分吸附体系中,Hb与吸附界面静电吸引作用强,吸附竞争力强。
3.建立了考虑吸附剂的粒径分布(PSD)的有效孔扩散模型(EPDM)、表面扩散模型(SDM)、Maxwell-Stefan模型(MSM)。通过蛋白质的间歇吸附动力学实验,研究了蛋白质(BSA)在阳离子交换剂(SP Sepharose FF)孔内的扩散。结果表明,这三种模型均能很好地拟合蛋白质的动态吸附曲线;但由于传质驱动力不同,各模型对孔内蛋白质沿吸附剂径向浓度分布图的预测存在较大差异。
4.利用共聚焦激光扫描显微镜(CLSM)直接观测了蛋白质在吸附剂颗粒内部的径向浓度分布情况。通过观测结果与考虑吸附剂PSD的模型(EPDM, SDM和MSM)拟合吸附剂孔内蛋白质径向浓度分布结果的比较,可以看出在目前的研究体系中SDM能够比EPDM更好地预测孔内蛋白质浓度分布图,MSM模型对孔内蛋白质浓度分布的预测结果介于另外两种模型之间。
5.由于在使用共聚焦显微镜观测吸附剂孔内蛋白质时,需要用荧光色素标
Ion-exchange equilibria and intraparticle mass transfer of protein in chromatography are of great importance for the design and optimization of bioseparation processes. Compared with the extensive application of ion-exchange chromatography in biotechnology, the mechanism and process analysis of ion-exchange system are still in the primary state. The details in this work are summarized as follows.
1. The adsorption equilibria of bovine serum albumin (BSA) and hemoglobin (Hb) to anion exchanger, DEAE Spherodex M, were studied by using statistical thermodynamic model (ST model). The ST model can well describe single and two-component protein equilibrium on the anion exchanger at different ionic strengths. The shielding effect of the co-ions in adsorption phase on the electrostatic interaction among protein molecules and adsorbents increases with an increase of ionic strength. At the same ionic strength, the electrostatic attraction between BSA molecules and DEAE Spherodex M is stronger, and the electrostatic repulsion among BSA molecules is stronger than that among Hb molecules. In two-component adsorption system, the electrostatic attraction between BSA molecules and adsorbent is stronger, BSA is more competitive. The effective protein charge values calculated respectively from ST model parameters 1/αiand k ii are consistent.
2. The adsorption equilibria of BSA and Hb to cation exchanger, SP Sepharose FF, were also studied by using ST model. The shielding effect of the co-ions in adsorption phase on the electrostatic interaction among protein molecules and adsorbents increases with increasing ionic strength. Moreover, the electrostatic interaction among protein molecules and adsorbents decreases with increasing pH. At the same pH values and ionic strengths, the electrostatic attraction between Hb molecules and SP Sepharose FF is stronger, and the electrostatic repulsion among Hb molecules is stronger than that among BSA molecules. In two-component adsorption system, the electrostatic attraction between Hb molecules and adsorbents is stronger, so Hb is more competitive.
3. The ion-exchange adsorption kinetics of BSA to the cation exchanger, SP Sepharose FF, was studied by batch adsorption experiments. Three diffusion models
引文
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