蛋白质在气/水界面上的物理化学性质和单分子膜成形
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摘要
蛋白质在气/水界面上的物理化学行为是软物质及软界面研究的热点之一，也是膜模拟化学的基础课题。它对于理解准二维空间的生物高分子凝聚态的现象和舰律具有重要的意义，对于使用“湿”的胶体化学方法来制备蛋白质功能材料具有指导意义。本文系统研究了蛋白质在气/水界面上的物理化学二个方面的问题：一、蛋白质自溶液向气/水界面的吸附动力学、吸附态分子构象及其变化规律；二、蛋白质在气/水界面上的铺展，蛋白质单分子膜的成形性能与规律，单分子膜成形过程中分子构象的变化及其规律。另外，对蛋白质稀溶液的粘度行为，分子流体力学尺寸、分子间作用力及其影响因素进行了研究。对蜘蛛丝的物理化学结构进行了表征和研究。
本文以天然蜘蛛丝蛋白、蚕丝蛋白和丝胶蛋白为蛋白质的模型物，以Wilhelmy片技术分别测定了三种蛋白质自稀溶液向表面吸附过程中，随时间的变化的表面压π值。运用Gibbs吸附等温式得到溶液吸附的表面浓度；利用蛋白质的π—t曲线，根据ln(dπ/dt)——π的关系曲线的二个不同的斜率，得到蛋白质不同吸附阶段在气/水界面上的分子截面积ΔA_1，ΔA_2，从而分析蛋白质的吸附机理。测定了蛋白质构象变化(改变溶液pH)对蛋白质吸附π值的影响，获得了蛋白质构象变化对吸附影响的规律。分别以丝胶蛋白和蚕丝蛋白为试样进行了球状蛋白和纤维状蛋白在气/水界面上的吸附性能的比较分析；同样，以蜘蛛丝蛋白和蚕丝蛋白为试样行了不同纤维状蛋白吸附性能的比较分析。
利用Langmuir膜槽进行的蛋白质单分子膜成形性能的研究，获得蜘蛛丝蛋白、蚕丝蛋白和丝胶蛋白三种不同蛋白质单分子膜的表面压π—分子面积A关
系曲线。以不同的蛋白水溶液为单分子膜成形的铺展剂，对蛋白质分子在亚相
水济液表面的分散和扩散条件以及不同的亚相水溶液条件，包括蛋白溶液浓度、
蛋白溶液的滴加体积、滴加方式、蛋白溶液的溶剂;亚相水溶液的pH，盐的种
类和浓度等对蛋白质单分子膜成形的影响进行了研究，得到了蛋白质单分子膜
的成形规律。由蛋白质单分子膜的二一A曲线获得气/液/固相变规律，得到不
同相态一F单分子膜中蛋白质分子构象结构的信息。由于亚相水溶液中的pH，盐
等较大地改变了蛋白质的构象从而改变了:一A曲线。对天然蜘蛛丝蛋白、蚕
丝蛋白和丝胶蛋白的单分子膜的成形性能进行了比较研究，根据不同蛋白质的
兀一A关系曲线的对比得到了球状蛋白和纤维状蛋白以及不同纤维状蛋白在气
/水界血_匕结构差异。
通过对丝胶蛋白的稀溶液的特性粘数〔几」的测定，获得稀溶液中蛋白质分
子流体力学尺寸的信息。根据Huggnins方程得到稀溶液中蛋白质分子间蛋白质
分子与溶剂分子相互作用参数KH。
利用氨基酸分析仪、红外光谱、扫描电子显微镜和原子力显微镜分别对蜘
蛛丝的氨基酸组成、蜘蛛丝蛋白的分子基团和分子构象结构、蜘蛛丝的表面形
态结构进行了表征。
研究结果表明，蛋白质的吸附过程由扩散控制的快速吸附阶段和分子排列、
取向的‘漫吸附速阶段所组成。中性水溶液中，水溶性的球状蛋白丝胶蛋白溶液
浓度0.0!25%至0.1%变化，吸附平衡表面压:值由10.ZmN/m变化至23.ZmN/m，
是相同条件卜纤维状蛋白蚕丝蛋白，蜘蛛丝蛋白的2倍左右。在这样的浓度范
围内，八A;在30一50.9人2范围内，而△AZ值的范围在230至570A2。蚕丝蛋白
的表面压二值在0.025%至0.1%范围内只达到6.2mN/m至12mN/m，与球形蛋
白丝胶蛋白相比，相同浓度下吸附速率低，表面吸附浓度较低。而吸附过程中
的△A:为32.8A2至67.4A2，△AZ值为462.8A2至866.7A2。由此可以认为纤维
状蛋白的吸附扩散速度低于球状蛋白，而其吸附分子的重排和取向后在界面上
的面积大于球状蛋白的占有面积。球状蛋白在气/水界面上的活性大于纤维状的
蛋白质。相同的溶液条件下，蜘蛛丝蛋白的表面压二值大于蚕丝。在浓度0.025%
至0.既范围内，△A，的值略低于蚕丝蛋白，而△AZ的值与蚕丝蛋白的值相近，
但高于丝胶蛋白的值。纤维状蛋白克服能垒后进入吸附层，分子重排所占面积
大于球形蛋白的面积。酸性条件下pH=3一4，pH二5，丝胶蛋白的表面压:值分
一2一
别为27.0mN/m和22.lmN/m，比pH=7分别增加7mN/m和ZmN/m，碱性条件
手’降低smN/m。pH值对纤维状蛋白的表面压:值影响较大，蚕丝蛋白表面压
:值比pH=7的增加值为20mN/m(PH=3 .5)，1 ZmN/m印H=5)，增加量超过pH==7
的1一2倍，蜘蛛丝蛋白的表面压:值的增量(对pH=7)也达18.4mN/m和
‘1.OmN/m。由此认为pH值对纤维状蛋白的吸附性能影响大于对球状蛋白的影响。
三种蛋白质的单分子膜的成形研究结果表明，蛋白质分子可以直接在亚相
水溶液上形成稳定的单分子膜。蜘蛛丝蛋白水溶液采用不同的成膜方式，滴加
法和亚相内注入法获得了基本相似的:一A曲线，证明了蜘蛛丝蛋白分子与带
有挥发性铺展溶剂的两性分子的成膜方式和机理的不同。前者的扩散成膜是依
靠蛋白质分子的吸附自由能在气/水界面上扩散吸附成膜;后者的成膜推动力源
自于挥发性溶剂的挥发带动成膜材料的扩散以及它的表面张力来形成单分子
The problem on physical and chemical behaviors of protein at air/water interface is a one of point of study on"soft mater and soft interface "and a basic subject of membrane-mimetic chemistry, which is significance in understanding the phenomena of condensed phase for biopolymers in quasi-two-dimensional space and also important for preparing the functional materials by use of the "aqueous methods" in colloid chemistry. The adsorption kinetics from the solution to the air/water interface and forming properties of protein monolayers at the interface have been investigated .The viscosity behavior of protein dilute solution, which includes the hydrodynamics molecular dimension of the protein ,intermolecular action force in the dilute solution and other factors, was also studied. Finally, the physicochemical structures of silk from spider Selenocosmia were characterized by means of IR, SEM and AFM. in this dissertation.
As protein sample in the experiment , spider protein, silk protein and sericin protein were used. The surface pressure of three proteins at air/water interface, which is cased by adsorption from the solution to the surface and then arranging and orienting at surface, were measured by means of Wilhelmy plate technique. Using Gibbs equation, the surface concentration of the solution can be evaluated. Analyzing the -t relationship of the protein adsorption and A1 and A2 given by 1n(d /dt)- relation curve, the adsorption mechanism for the protein may be predicted. The surface pressure value of the protein was changed with the change of molecular conformation in adsorption surface or the monolayer at air/water interface, which is cased by adding acid, or base, or salt to sub-phase solution. Moreover, the difference of absorption properties for two fibrous proteins, spider protein and silk protein, was analyzed and discussed, respectively.
The properties of monolayer forming for the proteins were investigated by using spreading technique on the surface of sub-phase in the Langmuir trough. The -A curve of the different monolayer of the proteins, including spider protein, silk protein and sericin protein, were obtained respectively. The formation conditions, e. g., the diffusion for the proteins at the surface of the solution, the sub-phase of the solution which includes it's pH value, the concentration, and the varieties salt and it's concentration in the solution and, the protein aqueous solution as spreading solution which it's spreading volume and spreading way are varied, have been investigated to determine the optimal processing conditions. The molecular conformation of the proteins varies with the sub-phase condition which is found by the change -A relationship curve for the protein monolayer. The molecular conformation in the protein monolayer was given by -A curve on which several transition points imply phase transitions representing gas/liquid/solid three phases in the monolayer. Comparing and the analyzing the properties of monolayer formation for three proteins, the differences of structure at air/water interface between the fibrous protein for spide protein and sild protein and globular protein for sericin protein nay be found on the -A relationship curve.
The intrinsic viscosity [ ]for the dilute proteins solution has been determined and the interaction coefficient between protein molecules, and between protein molecules and solvent maybe evaluated by using Huggins equation. The hydrodynamical dimension characterized by the intrinsic viscosity [ ] was important to know molecular conformations in the dilute solution.
The composition of amino acid, the molecular functional groups, the molecular conformation and the morphological structure of fibers for the spider silk were determined by using the analytical instrument for amino acid, IR, SEM and AFM, respectively.
The experiments results show that the protein adsorption process consists of two steps, of which one is rapid step controlled by diffusion of protein molecule from solution to surface, and the second st
本文以天然蜘蛛丝蛋白、蚕丝蛋白和丝胶蛋白为蛋白质的模型物，以Wilhelmy片技术分别测定了三种蛋白质自稀溶液向表面吸附过程中，随时间的变化的表面压π值。运用Gibbs吸附等温式得到溶液吸附的表面浓度；利用蛋白质的π—t曲线，根据ln(dπ/dt)——π的关系曲线的二个不同的斜率，得到蛋白质不同吸附阶段在气/水界面上的分子截面积ΔA_1，ΔA_2，从而分析蛋白质的吸附机理。测定了蛋白质构象变化(改变溶液pH)对蛋白质吸附π值的影响，获得了蛋白质构象变化对吸附影响的规律。分别以丝胶蛋白和蚕丝蛋白为试样进行了球状蛋白和纤维状蛋白在气/水界面上的吸附性能的比较分析；同样，以蜘蛛丝蛋白和蚕丝蛋白为试样行了不同纤维状蛋白吸附性能的比较分析。
利用Langmuir膜槽进行的蛋白质单分子膜成形性能的研究，获得蜘蛛丝蛋白、蚕丝蛋白和丝胶蛋白三种不同蛋白质单分子膜的表面压π—分子面积A关
系曲线。以不同的蛋白水溶液为单分子膜成形的铺展剂，对蛋白质分子在亚相
水济液表面的分散和扩散条件以及不同的亚相水溶液条件，包括蛋白溶液浓度、
蛋白溶液的滴加体积、滴加方式、蛋白溶液的溶剂;亚相水溶液的pH，盐的种
类和浓度等对蛋白质单分子膜成形的影响进行了研究，得到了蛋白质单分子膜
的成形规律。由蛋白质单分子膜的二一A曲线获得气/液/固相变规律，得到不
同相态一F单分子膜中蛋白质分子构象结构的信息。由于亚相水溶液中的pH，盐
等较大地改变了蛋白质的构象从而改变了:一A曲线。对天然蜘蛛丝蛋白、蚕
丝蛋白和丝胶蛋白的单分子膜的成形性能进行了比较研究，根据不同蛋白质的
兀一A关系曲线的对比得到了球状蛋白和纤维状蛋白以及不同纤维状蛋白在气
/水界血_匕结构差异。
通过对丝胶蛋白的稀溶液的特性粘数〔几」的测定，获得稀溶液中蛋白质分
子流体力学尺寸的信息。根据Huggnins方程得到稀溶液中蛋白质分子间蛋白质
分子与溶剂分子相互作用参数KH。
利用氨基酸分析仪、红外光谱、扫描电子显微镜和原子力显微镜分别对蜘
蛛丝的氨基酸组成、蜘蛛丝蛋白的分子基团和分子构象结构、蜘蛛丝的表面形
态结构进行了表征。
研究结果表明，蛋白质的吸附过程由扩散控制的快速吸附阶段和分子排列、
取向的‘漫吸附速阶段所组成。中性水溶液中，水溶性的球状蛋白丝胶蛋白溶液
浓度0.0!25%至0.1%变化，吸附平衡表面压:值由10.ZmN/m变化至23.ZmN/m，
是相同条件卜纤维状蛋白蚕丝蛋白，蜘蛛丝蛋白的2倍左右。在这样的浓度范
围内，八A;在30一50.9人2范围内，而△AZ值的范围在230至570A2。蚕丝蛋白
的表面压二值在0.025%至0.1%范围内只达到6.2mN/m至12mN/m，与球形蛋
白丝胶蛋白相比，相同浓度下吸附速率低，表面吸附浓度较低。而吸附过程中
的△A:为32.8A2至67.4A2，△AZ值为462.8A2至866.7A2。由此可以认为纤维
状蛋白的吸附扩散速度低于球状蛋白，而其吸附分子的重排和取向后在界面上
的面积大于球状蛋白的占有面积。球状蛋白在气/水界面上的活性大于纤维状的
蛋白质。相同的溶液条件下，蜘蛛丝蛋白的表面压二值大于蚕丝。在浓度0.025%
至0.既范围内，△A，的值略低于蚕丝蛋白，而△AZ的值与蚕丝蛋白的值相近，
但高于丝胶蛋白的值。纤维状蛋白克服能垒后进入吸附层，分子重排所占面积
大于球形蛋白的面积。酸性条件下pH=3一4，pH二5，丝胶蛋白的表面压:值分
一2一
别为27.0mN/m和22.lmN/m，比pH=7分别增加7mN/m和ZmN/m，碱性条件
手’降低smN/m。pH值对纤维状蛋白的表面压:值影响较大，蚕丝蛋白表面压
:值比pH=7的增加值为20mN/m(PH=3 .5)，1 ZmN/m印H=5)，增加量超过pH==7
的1一2倍，蜘蛛丝蛋白的表面压:值的增量(对pH=7)也达18.4mN/m和
‘1.OmN/m。由此认为pH值对纤维状蛋白的吸附性能影响大于对球状蛋白的影响。
三种蛋白质的单分子膜的成形研究结果表明，蛋白质分子可以直接在亚相
水溶液上形成稳定的单分子膜。蜘蛛丝蛋白水溶液采用不同的成膜方式，滴加
法和亚相内注入法获得了基本相似的:一A曲线，证明了蜘蛛丝蛋白分子与带
有挥发性铺展溶剂的两性分子的成膜方式和机理的不同。前者的扩散成膜是依
靠蛋白质分子的吸附自由能在气/水界面上扩散吸附成膜;后者的成膜推动力源
自于挥发性溶剂的挥发带动成膜材料的扩散以及它的表面张力来形成单分子
The problem on physical and chemical behaviors of protein at air/water interface is a one of point of study on"soft mater and soft interface "and a basic subject of membrane-mimetic chemistry, which is significance in understanding the phenomena of condensed phase for biopolymers in quasi-two-dimensional space and also important for preparing the functional materials by use of the "aqueous methods" in colloid chemistry. The adsorption kinetics from the solution to the air/water interface and forming properties of protein monolayers at the interface have been investigated .The viscosity behavior of protein dilute solution, which includes the hydrodynamics molecular dimension of the protein ,intermolecular action force in the dilute solution and other factors, was also studied. Finally, the physicochemical structures of silk from spider Selenocosmia were characterized by means of IR, SEM and AFM. in this dissertation.
As protein sample in the experiment , spider protein, silk protein and sericin protein were used. The surface pressure of three proteins at air/water interface, which is cased by adsorption from the solution to the surface and then arranging and orienting at surface, were measured by means of Wilhelmy plate technique. Using Gibbs equation, the surface concentration of the solution can be evaluated. Analyzing the -t relationship of the protein adsorption and A1 and A2 given by 1n(d /dt)- relation curve, the adsorption mechanism for the protein may be predicted. The surface pressure value of the protein was changed with the change of molecular conformation in adsorption surface or the monolayer at air/water interface, which is cased by adding acid, or base, or salt to sub-phase solution. Moreover, the difference of absorption properties for two fibrous proteins, spider protein and silk protein, was analyzed and discussed, respectively.
The properties of monolayer forming for the proteins were investigated by using spreading technique on the surface of sub-phase in the Langmuir trough. The -A curve of the different monolayer of the proteins, including spider protein, silk protein and sericin protein, were obtained respectively. The formation conditions, e. g., the diffusion for the proteins at the surface of the solution, the sub-phase of the solution which includes it's pH value, the concentration, and the varieties salt and it's concentration in the solution and, the protein aqueous solution as spreading solution which it's spreading volume and spreading way are varied, have been investigated to determine the optimal processing conditions. The molecular conformation of the proteins varies with the sub-phase condition which is found by the change -A relationship curve for the protein monolayer. The molecular conformation in the protein monolayer was given by -A curve on which several transition points imply phase transitions representing gas/liquid/solid three phases in the monolayer. Comparing and the analyzing the properties of monolayer formation for three proteins, the differences of structure at air/water interface between the fibrous protein for spide protein and sild protein and globular protein for sericin protein nay be found on the -A relationship curve.
The intrinsic viscosity [ ]for the dilute proteins solution has been determined and the interaction coefficient between protein molecules, and between protein molecules and solvent maybe evaluated by using Huggins equation. The hydrodynamical dimension characterized by the intrinsic viscosity [ ] was important to know molecular conformations in the dilute solution.
The composition of amino acid, the molecular functional groups, the molecular conformation and the morphological structure of fibers for the spider silk were determined by using the analytical instrument for amino acid, IR, SEM and AFM, respectively.
The experiments results show that the protein adsorption process consists of two steps, of which one is rapid step controlled by diffusion of protein molecule from solution to surface, and the second st
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