液-固界面的吸附及溶菌酶在液相色谱中复性的应用研究
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摘要
基因重组蛋白的复性问题是生物工程下游技术中的一个瓶颈。蛋白折叠色谱是近年来出现的一个新的复性方法，由于它能显著地改进复性产率，而且能将复性与分离纯化合为一步，降低下游成本，所以是当前生物技术中的一个研究热点。由于液相色谱中固定相与流动相之间的界面，即液-固界面对蛋白折叠起着至关重要的作用。所以本文将首先从描述液-固界面的模型入手，研究溶质在液-固界面的吸附行为受到的影响，在此基础上对变性蛋白在离子交换色谱(IEC)，疏水色谱(HIC)及排阻色谱(SEC)中的复性进行研究。
论文包括以下八个部分：
1．文献综述：界面吸附广泛存在于自然界中，而其中液-固吸附比气-固吸附要复杂的多和重要的多。本文对研究液-固吸附的意义及液-固吸附的特点、难点之所在，以及目前液-固吸附理论的研究进展进行了综述。蛋白折叠的研究具有重大的理论意义和重要的实际价值，目前的研究还一直处于经验摸索的阶段。本文从基本的热力学假说、动力学控制入手，提出了蛋白复性的基本策略，对在这些基本策略指引下发展出的各种复性方法及进展进行了全面地综述。共包括文献172篇。
2．以计量置换吸附理论(stoichiometric displacement theory for adsorption，SDT-A)为基础，从理论上首次推导出一个新的表征溶剂浓度对吸附量影响的吸附等温线。运用不同的假设条件和简化，得到了含有两参数，三参数和四参数的方程。两参数方程适用于溶质浓度非常低的情况，吸附量与溶剂浓度之间是一个线性的对数关系。三参数和四参数方程则适用于溶质浓度较大的情况，是一种非线性的对数关系。用芳香醇同系物在反相液相色谱(RPLC)中的前沿分析法进行了验证，新的方程都能很好的拟合实验数据。
3．以SDT为基础，首次从理论上推导出计量吸附理论中表征溶质对固定相亲合势大小的参数β_α值与流动相中强置换剂浓度的对数呈线性关系，并用苯的衍生物进行了实验验证，获得了满意的结果。而且，计量置换参数Z的分量是两个很有用的参数，但一直没有从RPLC中单独得到过，从新推导的这个吸附亲合势与溶剂浓度之间的方程式很容易得到Z的分量n和q值。从理论上首次将SDT-A和计量置换保留理论(stoichiometric displacement theory for retention，
SDT一R)这两个分别表征吸附和保留过程的理论统一了起来，证明在SDT一R
中的容量因子的对数logk’与SDT一A中溶质对吸附剂亲合势的参数刀。之间存
在线性关系。从理论上推导出刀。值与溶剂浓度的对数作图收敛点的存在并阐
明了收敛点坐标的物理意义，与实验作图法得到的数据进行了对比，符合情
况很好。
4.从气一固吸附体系中推导出的Langmuir方程，在气一固吸附中是一个定量的公
式，但近一世纪来只能经验性地描述组份在液相中的吸附。本研究以液一固界
面上的SDT为基础，考虑到液一固吸附体系中各组分之间的相互作用，首次从
理论上推导出了在液一固体系中描述在不同溶剂浓度条件下，溶质从溶液中吸
附的Langmuir公式，并称其为扩展的Langmuir公式。以不同溶剂浓度条件
下所得到的吸附等温线数据对理论推导出的扩展的Langmuir公式进行了验
证，与计量置换平衡中的参数相关联，表明用吸附等温线法计算的计量置换
参数Z与用高效液相色谱法得到的Z值符合程度很好.同时研究发现卵清蛋
白在疏水整体色谱柱的吸附与流动相中盐浓度有直接的关系。当蛋白在色谱
柱上保留时，在相同的溶质浓度条件下其吸附量的对数与置换剂水的摩尔浓
度对数之间有非常好的线性关系。
5.以溶菌酶(切s)为目标蛋白，用紫外(UV)，荧光(FR)，RPLC，弱阳离子交换
色谱(WCX)及SDT中的Z值系统地研究了四种不同变性状态(肌变还原与
非还原，脉变还原与非还原)的切s的分子构象变化，并与其天然态进行了比
较.发现这四种不同变性状态的切s与天然态Lys的uv之间没有明显区别，
但与天然态相比，这四种变性态的场S的FR却都有红移现象.然而荧光光谱
并不能反映出这四种变性态以不同的变性方式所引起的分子构象变化.Lys在
RPLC和WCX的保留行为却能够反映出其还原变性态与非还原变性态之间
的差别，但仍不能显示出不同变性剂引起的蛋白分子的构象变化.只有液一固
界面上SDT中的参数Z值才能反映出上述所有的差别.还引入了变性度和相
对变性度的概念以定量地方式表征在不同变性环境中切s分子构象变化的程
度。
6.首次用Hlc复性了还原变性的琢S。研究了还原变性琢S在三种疏水性不同
的HIC柱上的复性情况。发现其在疏水性最弱的XDM一GMI型色谱柱的复性
效率最高，蛋白浓度为2.omg/mL时可以达到90%以上，但在更高的蛋白浓
度时其复性效率显著降低。发现在疏水性更弱的WCX柱上，还原变性切s
的保留呈现离子交换和疏水双保留机理。研究了还原变性切s在此wcx柱
用疏水模式复性的情况，发现在这种WCX柱上用疏水和离子交换双梯度进
行复性可以明显地提高其复性效率，当蛋白浓度达到17.2m酬mL时，活性回
收率达到80%。同时考察了流动相组成，脉
Protein folding remains a bottle-neck issues in the down-stream technology in biotechnology. As a new method, refolding chromatography can improve the bioactivity yield and simultaneously accomplish its purification, so it has been paid much attention in recent years. The interface of liquid-solid in liquid chromatography (LC) gives main contribution to protein refolding, so the impacts of the solute adsorption in the liquid-solid systems were investigated firstly. Then the refolding of protein in the ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) was studied. The thesis includes eight parts as the follows:
1. Review: Interfacial phenomena are encountered broadly in the nature. Adsorption at the interface of liquid-solid systems is more complex and important than that of gas-solid systems. It was reviewed about the importance of studying the adsorption in liquid-solid systems, and specialty and difficulty of the liquid-solid interface and development of recently theoretical studies. Protein folding is a subject of fundamental and practical importance. Though much effort has been expended to solve this problem, no universal method has been established. From the thermodynamic hypothesis and kinetic controlling, a fundamental refolding strategy was introduced. All kinds of method and development based on the strategy were reviewed. 172 references were listed.
2. Based on the stoichiometric displacement theory for adsorption (SDT-A), a new adsorption isotherm that relates to the amount of solute adsorbed to the solvent concentration in bulk solution is firstly proposed. The obtained equation may be simplified to an expression containing two, three, and four parameters according to different assumptions. The equation with two parameters, valid for low concentrations of solute, is a log-log linear relationship. The equations with three and four parameters are valid as the adsorbed amounts are larger and show non-linear logarithmic relationships. Tests with a homologous series of aromatic alcohols by frontal analysis (FA) in reversed-phase liquid chromatography (RPLC)
demonstrate that experimental results fit those equations well.
3. Based on the SDT-A of solute, an equation expressing the linear relationship between the affinity of component to adsorbent, Ba, and the logarithm of the molar concentration of solvent in the bulk solution employed, logaD was derived. The derived equation was tested by the derivatives of benzene under different methanol concentrations by FA of RPLC. A satisfactory result was obtained. Morever, the n and q terms' values (moles of the solvent separately released from the adsorbent and solute, respectively, as one mole of solute is adsorbed) which are the fractions of the stoichiometric parameter Z (Z- n +q), are very useful but hard to obtain only by RPLC alone. However, both were obtained from this relationship and tested with the presented method. The SDT-A, describing the adsorption process, and the SDM-R, describing the retention process, were firstly unified theoretically. It is proved that there exists a linear relationship between the logarithm of capacity factor logA:' in SRT-R and Bain SDT-A. The expression of the concentration convergent point of the homologue was further derived and validated.
4. The Langmuir equation, originally derived for gas-solid system and only a quantitative equation in this system, has been widely used to describe empirically solute adsorption from solution for almost a century. In this research, based on various interactions among components in liquid-solid adsorbed system, and by using the SDT-A, the Langmuir equation for solute adsorption from a liquid-solid adsorbed system was firstly theoretically derived and called as Extended Langmuir equation. The parameters in the Extended Langmuir equation were related to that of the stoichiometric displacement equilibrium, thus the former can be extended to describe solute adsorption under v
论文包括以下八个部分：
1．文献综述：界面吸附广泛存在于自然界中，而其中液-固吸附比气-固吸附要复杂的多和重要的多。本文对研究液-固吸附的意义及液-固吸附的特点、难点之所在，以及目前液-固吸附理论的研究进展进行了综述。蛋白折叠的研究具有重大的理论意义和重要的实际价值，目前的研究还一直处于经验摸索的阶段。本文从基本的热力学假说、动力学控制入手，提出了蛋白复性的基本策略，对在这些基本策略指引下发展出的各种复性方法及进展进行了全面地综述。共包括文献172篇。
2．以计量置换吸附理论(stoichiometric displacement theory for adsorption，SDT-A)为基础，从理论上首次推导出一个新的表征溶剂浓度对吸附量影响的吸附等温线。运用不同的假设条件和简化，得到了含有两参数，三参数和四参数的方程。两参数方程适用于溶质浓度非常低的情况，吸附量与溶剂浓度之间是一个线性的对数关系。三参数和四参数方程则适用于溶质浓度较大的情况，是一种非线性的对数关系。用芳香醇同系物在反相液相色谱(RPLC)中的前沿分析法进行了验证，新的方程都能很好的拟合实验数据。
3．以SDT为基础，首次从理论上推导出计量吸附理论中表征溶质对固定相亲合势大小的参数β_α值与流动相中强置换剂浓度的对数呈线性关系，并用苯的衍生物进行了实验验证，获得了满意的结果。而且，计量置换参数Z的分量是两个很有用的参数，但一直没有从RPLC中单独得到过，从新推导的这个吸附亲合势与溶剂浓度之间的方程式很容易得到Z的分量n和q值。从理论上首次将SDT-A和计量置换保留理论(stoichiometric displacement theory for retention，
SDT一R)这两个分别表征吸附和保留过程的理论统一了起来，证明在SDT一R
中的容量因子的对数logk’与SDT一A中溶质对吸附剂亲合势的参数刀。之间存
在线性关系。从理论上推导出刀。值与溶剂浓度的对数作图收敛点的存在并阐
明了收敛点坐标的物理意义，与实验作图法得到的数据进行了对比，符合情
况很好。
4.从气一固吸附体系中推导出的Langmuir方程，在气一固吸附中是一个定量的公
式，但近一世纪来只能经验性地描述组份在液相中的吸附。本研究以液一固界
面上的SDT为基础，考虑到液一固吸附体系中各组分之间的相互作用，首次从
理论上推导出了在液一固体系中描述在不同溶剂浓度条件下，溶质从溶液中吸
附的Langmuir公式，并称其为扩展的Langmuir公式。以不同溶剂浓度条件
下所得到的吸附等温线数据对理论推导出的扩展的Langmuir公式进行了验
证，与计量置换平衡中的参数相关联，表明用吸附等温线法计算的计量置换
参数Z与用高效液相色谱法得到的Z值符合程度很好.同时研究发现卵清蛋
白在疏水整体色谱柱的吸附与流动相中盐浓度有直接的关系。当蛋白在色谱
柱上保留时，在相同的溶质浓度条件下其吸附量的对数与置换剂水的摩尔浓
度对数之间有非常好的线性关系。
5.以溶菌酶(切s)为目标蛋白，用紫外(UV)，荧光(FR)，RPLC，弱阳离子交换
色谱(WCX)及SDT中的Z值系统地研究了四种不同变性状态(肌变还原与
非还原，脉变还原与非还原)的切s的分子构象变化，并与其天然态进行了比
较.发现这四种不同变性状态的切s与天然态Lys的uv之间没有明显区别，
但与天然态相比，这四种变性态的场S的FR却都有红移现象.然而荧光光谱
并不能反映出这四种变性态以不同的变性方式所引起的分子构象变化.Lys在
RPLC和WCX的保留行为却能够反映出其还原变性态与非还原变性态之间
的差别，但仍不能显示出不同变性剂引起的蛋白分子的构象变化.只有液一固
界面上SDT中的参数Z值才能反映出上述所有的差别.还引入了变性度和相
对变性度的概念以定量地方式表征在不同变性环境中切s分子构象变化的程
度。
6.首次用Hlc复性了还原变性的琢S。研究了还原变性琢S在三种疏水性不同
的HIC柱上的复性情况。发现其在疏水性最弱的XDM一GMI型色谱柱的复性
效率最高，蛋白浓度为2.omg/mL时可以达到90%以上，但在更高的蛋白浓
度时其复性效率显著降低。发现在疏水性更弱的WCX柱上，还原变性切s
的保留呈现离子交换和疏水双保留机理。研究了还原变性切s在此wcx柱
用疏水模式复性的情况，发现在这种WCX柱上用疏水和离子交换双梯度进
行复性可以明显地提高其复性效率，当蛋白浓度达到17.2m酬mL时，活性回
收率达到80%。同时考察了流动相组成，脉
Protein folding remains a bottle-neck issues in the down-stream technology in biotechnology. As a new method, refolding chromatography can improve the bioactivity yield and simultaneously accomplish its purification, so it has been paid much attention in recent years. The interface of liquid-solid in liquid chromatography (LC) gives main contribution to protein refolding, so the impacts of the solute adsorption in the liquid-solid systems were investigated firstly. Then the refolding of protein in the ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) was studied. The thesis includes eight parts as the follows:
1. Review: Interfacial phenomena are encountered broadly in the nature. Adsorption at the interface of liquid-solid systems is more complex and important than that of gas-solid systems. It was reviewed about the importance of studying the adsorption in liquid-solid systems, and specialty and difficulty of the liquid-solid interface and development of recently theoretical studies. Protein folding is a subject of fundamental and practical importance. Though much effort has been expended to solve this problem, no universal method has been established. From the thermodynamic hypothesis and kinetic controlling, a fundamental refolding strategy was introduced. All kinds of method and development based on the strategy were reviewed. 172 references were listed.
2. Based on the stoichiometric displacement theory for adsorption (SDT-A), a new adsorption isotherm that relates to the amount of solute adsorbed to the solvent concentration in bulk solution is firstly proposed. The obtained equation may be simplified to an expression containing two, three, and four parameters according to different assumptions. The equation with two parameters, valid for low concentrations of solute, is a log-log linear relationship. The equations with three and four parameters are valid as the adsorbed amounts are larger and show non-linear logarithmic relationships. Tests with a homologous series of aromatic alcohols by frontal analysis (FA) in reversed-phase liquid chromatography (RPLC)
demonstrate that experimental results fit those equations well.
3. Based on the SDT-A of solute, an equation expressing the linear relationship between the affinity of component to adsorbent, Ba, and the logarithm of the molar concentration of solvent in the bulk solution employed, logaD was derived. The derived equation was tested by the derivatives of benzene under different methanol concentrations by FA of RPLC. A satisfactory result was obtained. Morever, the n and q terms' values (moles of the solvent separately released from the adsorbent and solute, respectively, as one mole of solute is adsorbed) which are the fractions of the stoichiometric parameter Z (Z- n +q), are very useful but hard to obtain only by RPLC alone. However, both were obtained from this relationship and tested with the presented method. The SDT-A, describing the adsorption process, and the SDM-R, describing the retention process, were firstly unified theoretically. It is proved that there exists a linear relationship between the logarithm of capacity factor logA:' in SRT-R and Bain SDT-A. The expression of the concentration convergent point of the homologue was further derived and validated.
4. The Langmuir equation, originally derived for gas-solid system and only a quantitative equation in this system, has been widely used to describe empirically solute adsorption from solution for almost a century. In this research, based on various interactions among components in liquid-solid adsorbed system, and by using the SDT-A, the Langmuir equation for solute adsorption from a liquid-solid adsorbed system was firstly theoretically derived and called as Extended Langmuir equation. The parameters in the Extended Langmuir equation were related to that of the stoichiometric displacement equilibrium, thus the former can be extended to describe solute adsorption under v
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