几种低分子量有机酸、磷酸对BSA和酶在土壤胶体、矿物表面的吸附及活性影响
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摘要
论文在分析总结了国内外有关研究的基础上，用批量法和傅立叶变换红外光谱(FTIR)法首次系统地研究了乙酸、草酸、酒石酸和磷酸几种配体的浓度、pH及加入顺序对牛血清白蛋白(BSA)、酸性磷酸酶在黄棕壤、砖红壤、高岭石、针铁矿和二氧化锰表面的吸附行为及活性的影响，探讨了这些配体对蛋白吸附机理及二级结构的影响。这对深入探讨土壤对酶与白蛋白吸附中有关组分的相互作用机理，阐明影响土壤酶活性的因素，对土壤肥力和环境质量的提高与调控有重要的理论和实际意义。取得的主要结果和进展有：
1、在FTIR测试中，首次用真空冷冻干燥法明确了：草酸和针铁矿表面均可使BSA的正常β-折叠减少，无规卷曲结构增加；两者对BSA的异常β-折叠、α-螺旋和β-转角结构含量的贡献相反，针铁矿表面可使有序结构增加且与蛋白加入浓度密切相关：草酸可使BSA中的疏水性羰基增加，亲水性羰基减少，而针铁矿表面的作用则相反。
2、提出了：1)对供试胶体悬浊液中土壤胶体进行快速精确定量的方法，即：将制成的供试胶体悬浊液经超声波和磁力搅拌充分分散，再用移液枪定量吸取的方法是可行的；2)在待试液混合前分别调控其pH以控制试验体系pH的方法，可取得较为满意的结果。
3、不同阴离子配体对酶与蛋白在土壤胶体、矿物表面的吸附及活性影响研究中，首次明确了配体对蛋白吸附及活性的影响程度取决于胶体的表面类型、酶与蛋白的种类、配体分子的大小、浓度、功能团的数量、体系的pH及配体的加入顺序等。
1)首次揭示出：＜10mmol／L的乙酸对供试胶体表面蛋白的吸附有促进作用，＞10mmol／L的乙酸则轻微抑制；＜5mmol／L的草酸对蛋白吸附表现为抑制，＞5mmol／L后有促进作用；酒石酸或磷酸浓度的升高对蛋白吸附表现为一直抑制。磷酸对针铁矿上蛋白吸附抑制最显著，酒石酸对土壤胶体和高岭石上的抑制最明显，且发现不同阴离子配体的存在使供试土壤胶体和矿物对蛋白的吸附产生选择性，揭示出胶体矿物的表面类型、配体分子的大小及功能团在决定胶体矿物表面蛋白吸附中起重要作用。
2)阐明了：A)蛋白等温吸附曲线的类型与配体的种类、浓度密切相关；B)乙酸、草酸、低浓度酒石酸和磷酸体系中，蛋白的等温吸附曲线与Langmuir方程(类型为H或L型)相符，而高浓度酒石酸(＞50mmol／L)或磷酸(＞100mmol／L)体系中的蛋白等温吸附符合线性方程(C型)；C)乙酸或磷酸加入顺序对供试胶体酶的等温吸附曲线形状、最大吸附量和吸附结合能影响较小(磷酸对针铁矿除外)；草酸或酒石酸对上述吸附参数影响均显著。这可能与它们加入顺序不同导致的吸附机理不同有关。
3)发现：A)胶体表面类型(硅氧烷表面或羟基化表面)及阴离子配体对胶体表面负电荷的贡献量在酶的吸附及活性保持中的重要性：B)阴离子配体后加入时酶的吸附量一般比配体先加入时的高；C)配体加入顺序对二氧化锰的影响最显著，对砖红壤和
高岭石的居中;对针铁矿的次之:对黄棕壤的最小。D)乙酸或草酸加入顺序对高岭
石上固定化酶比活性的影响最大，而酒石酸或磷酸则对针铁矿影响最大。
4)供试胶体上酶的最大吸附量的pH一般在酶的等电点和供试胶体的PZC之间，
曲线形状为“坡形”或“钟形”，本试验结果完善并证实了非对称性模型假说。随配体
体系pH变化，针铁矿上酶的比活性远比其它胶体上的高，且一般有两个最大比活性点，
分别在pH 4.0和6.5附近;其它胶体上则只有一个，在pH 4.8一6.1，与原酶相比有所漂
移，说明配体的存在使固定化酶活性中心的微域环境发生改变。
5)供试胶体与不同配体复合物(L一C)的无机催化活性大小顺序为:磷酸>>酒石酸
>草酸>乙酸，这与文献报道的有关配位复合物的稳定常数及其对供试胶体表面的溶解能
力非常吻合。从胶体类型来看则为:针铁矿>>二氧化锰>层状硅酸盐类矿物，说明配体
在胶体表面的存在使胶体具有了伪酶的作用。
The effect of the concentration, pH of several ligands such as acetate, oxalate, tartrate and phosphate solution and their orders added on the adsorption and activity of BSA and acid phosphatase on yellow brown soil, latosol, kaolinite, goethite and -MnO2 was first systematically studied by using batch and FTIR methods after analyzed and studied the relative literature that reported in the world. The impact of these ligands on the adsorption mechanism and secondary structure of protein was simultaneously engaged. These studies have important significances in scientific field and practice for probe into interaction mechanism of the relate compositions in the adsorption of BSA and enzyme and for clarification the factors affecting soil enzyme activity, as well as for improving soil fertility and environmental quality. Some main results and progressions are illustrated as follows:
1. The results of FTIR in vacuum for the first time indicated that: Both oxalate and goethite surface can make the -sheet of BSA decrease, and make that of random bent increase of BSA. The effect action of oxalate and goethite surface on the content of abnormity -sheet, -helix and -turn structure in BSA was contrary, whereas the goethite surface can make the ordered structure increase of BSA, and it related nearly to the concentration of BSA added into the system. Oxalate can make the increase of C=O in hydrophobic situation of BSA, and make the decrease of C=O in polar situation of BSA. However, the action of goethite surface in this aspect was contrary.
2. The quick-speed and accurate method provided by author such as 1). Suspension prepared by adding deionized water into soil colloids and minerals tested to be dispersed by ultrasonic and magnetic force stirrer, sucked with finnpipette to get quantitative soil colloids and minerals was validity; 2). The method by adjusting the pH of each sample solution before mixing them can control the pH of reaction system in the true sense.
3. In the research of effects of ligands on adsorption and activity of protein and enzyme on soil colloids and minerals studied, we elucidated for the first time that: The influencing degree of ligands on adsorption and activity of protein and enzyme were depended on surface type of colloids, the kind of protein and enzyme, the magnitude ligand, the content of function group, the concentration and pH of lignad solution, and the addition order of ligands and enzyme and so on.
1) First revealed that: (1) there were stimulative action of acetate in low concentration
(<10mmol/L) for proteins adsorption on the surface of soil colloids and minerals studied whereas the weak restraint action would be happened when acetate concentration was over 10 mmol/L; (2) For oxalate system, the adsorption amount of protein firstly decreased (0-5mmol/L), followed with an increment (>5mmol/L); (3) For the tartrate and phosphate system, with the concentration of them increasing, the amount of protein adsorbed by colloids tested would be depressed considerably and progressively. (4) Phosphate shows the greatest impact for the adsorption of protein on Fe oxide surface whereas the tartrate may pose a greater effect on the protein adsorption for other sorts of surfaces, such as layer aluminum silicate minerals. One found that the selectivity of soil colloids and minerals for the protein adsorption would be caused by the presence of different anion ligands in system. This proved that the protein adsorption on the surface of soil colloids and minerals at a certain degree depend upon the surface type of colloids, the molecular magnitude and function group of ligands.
2). Elucidated that: (1) The type of adsorption isotherm related nearly to the kind and concentration of ligands. (2) In acetate, oxalate and low concentrations of tartrate and phosphate solution, the adsorption isotherms for the proteins on soil colloids and minerals tested conformed to the Langmuir model (L or H type) whereas a linear (C type) adsorption isotherm was obtained at higher concentrations of ta
1、在FTIR测试中，首次用真空冷冻干燥法明确了：草酸和针铁矿表面均可使BSA的正常β-折叠减少，无规卷曲结构增加；两者对BSA的异常β-折叠、α-螺旋和β-转角结构含量的贡献相反，针铁矿表面可使有序结构增加且与蛋白加入浓度密切相关：草酸可使BSA中的疏水性羰基增加，亲水性羰基减少，而针铁矿表面的作用则相反。
2、提出了：1)对供试胶体悬浊液中土壤胶体进行快速精确定量的方法，即：将制成的供试胶体悬浊液经超声波和磁力搅拌充分分散，再用移液枪定量吸取的方法是可行的；2)在待试液混合前分别调控其pH以控制试验体系pH的方法，可取得较为满意的结果。
3、不同阴离子配体对酶与蛋白在土壤胶体、矿物表面的吸附及活性影响研究中，首次明确了配体对蛋白吸附及活性的影响程度取决于胶体的表面类型、酶与蛋白的种类、配体分子的大小、浓度、功能团的数量、体系的pH及配体的加入顺序等。
1)首次揭示出：＜10mmol／L的乙酸对供试胶体表面蛋白的吸附有促进作用，＞10mmol／L的乙酸则轻微抑制；＜5mmol／L的草酸对蛋白吸附表现为抑制，＞5mmol／L后有促进作用；酒石酸或磷酸浓度的升高对蛋白吸附表现为一直抑制。磷酸对针铁矿上蛋白吸附抑制最显著，酒石酸对土壤胶体和高岭石上的抑制最明显，且发现不同阴离子配体的存在使供试土壤胶体和矿物对蛋白的吸附产生选择性，揭示出胶体矿物的表面类型、配体分子的大小及功能团在决定胶体矿物表面蛋白吸附中起重要作用。
2)阐明了：A)蛋白等温吸附曲线的类型与配体的种类、浓度密切相关；B)乙酸、草酸、低浓度酒石酸和磷酸体系中，蛋白的等温吸附曲线与Langmuir方程(类型为H或L型)相符，而高浓度酒石酸(＞50mmol／L)或磷酸(＞100mmol／L)体系中的蛋白等温吸附符合线性方程(C型)；C)乙酸或磷酸加入顺序对供试胶体酶的等温吸附曲线形状、最大吸附量和吸附结合能影响较小(磷酸对针铁矿除外)；草酸或酒石酸对上述吸附参数影响均显著。这可能与它们加入顺序不同导致的吸附机理不同有关。
3)发现：A)胶体表面类型(硅氧烷表面或羟基化表面)及阴离子配体对胶体表面负电荷的贡献量在酶的吸附及活性保持中的重要性：B)阴离子配体后加入时酶的吸附量一般比配体先加入时的高；C)配体加入顺序对二氧化锰的影响最显著，对砖红壤和
高岭石的居中;对针铁矿的次之:对黄棕壤的最小。D)乙酸或草酸加入顺序对高岭
石上固定化酶比活性的影响最大，而酒石酸或磷酸则对针铁矿影响最大。
4)供试胶体上酶的最大吸附量的pH一般在酶的等电点和供试胶体的PZC之间，
曲线形状为“坡形”或“钟形”，本试验结果完善并证实了非对称性模型假说。随配体
体系pH变化，针铁矿上酶的比活性远比其它胶体上的高，且一般有两个最大比活性点，
分别在pH 4.0和6.5附近;其它胶体上则只有一个，在pH 4.8一6.1，与原酶相比有所漂
移，说明配体的存在使固定化酶活性中心的微域环境发生改变。
5)供试胶体与不同配体复合物(L一C)的无机催化活性大小顺序为:磷酸>>酒石酸
>草酸>乙酸，这与文献报道的有关配位复合物的稳定常数及其对供试胶体表面的溶解能
力非常吻合。从胶体类型来看则为:针铁矿>>二氧化锰>层状硅酸盐类矿物，说明配体
在胶体表面的存在使胶体具有了伪酶的作用。
The effect of the concentration, pH of several ligands such as acetate, oxalate, tartrate and phosphate solution and their orders added on the adsorption and activity of BSA and acid phosphatase on yellow brown soil, latosol, kaolinite, goethite and -MnO2 was first systematically studied by using batch and FTIR methods after analyzed and studied the relative literature that reported in the world. The impact of these ligands on the adsorption mechanism and secondary structure of protein was simultaneously engaged. These studies have important significances in scientific field and practice for probe into interaction mechanism of the relate compositions in the adsorption of BSA and enzyme and for clarification the factors affecting soil enzyme activity, as well as for improving soil fertility and environmental quality. Some main results and progressions are illustrated as follows:
1. The results of FTIR in vacuum for the first time indicated that: Both oxalate and goethite surface can make the -sheet of BSA decrease, and make that of random bent increase of BSA. The effect action of oxalate and goethite surface on the content of abnormity -sheet, -helix and -turn structure in BSA was contrary, whereas the goethite surface can make the ordered structure increase of BSA, and it related nearly to the concentration of BSA added into the system. Oxalate can make the increase of C=O in hydrophobic situation of BSA, and make the decrease of C=O in polar situation of BSA. However, the action of goethite surface in this aspect was contrary.
2. The quick-speed and accurate method provided by author such as 1). Suspension prepared by adding deionized water into soil colloids and minerals tested to be dispersed by ultrasonic and magnetic force stirrer, sucked with finnpipette to get quantitative soil colloids and minerals was validity; 2). The method by adjusting the pH of each sample solution before mixing them can control the pH of reaction system in the true sense.
3. In the research of effects of ligands on adsorption and activity of protein and enzyme on soil colloids and minerals studied, we elucidated for the first time that: The influencing degree of ligands on adsorption and activity of protein and enzyme were depended on surface type of colloids, the kind of protein and enzyme, the magnitude ligand, the content of function group, the concentration and pH of lignad solution, and the addition order of ligands and enzyme and so on.
1) First revealed that: (1) there were stimulative action of acetate in low concentration
(<10mmol/L) for proteins adsorption on the surface of soil colloids and minerals studied whereas the weak restraint action would be happened when acetate concentration was over 10 mmol/L; (2) For oxalate system, the adsorption amount of protein firstly decreased (0-5mmol/L), followed with an increment (>5mmol/L); (3) For the tartrate and phosphate system, with the concentration of them increasing, the amount of protein adsorbed by colloids tested would be depressed considerably and progressively. (4) Phosphate shows the greatest impact for the adsorption of protein on Fe oxide surface whereas the tartrate may pose a greater effect on the protein adsorption for other sorts of surfaces, such as layer aluminum silicate minerals. One found that the selectivity of soil colloids and minerals for the protein adsorption would be caused by the presence of different anion ligands in system. This proved that the protein adsorption on the surface of soil colloids and minerals at a certain degree depend upon the surface type of colloids, the molecular magnitude and function group of ligands.
2). Elucidated that: (1) The type of adsorption isotherm related nearly to the kind and concentration of ligands. (2) In acetate, oxalate and low concentrations of tartrate and phosphate solution, the adsorption isotherms for the proteins on soil colloids and minerals tested conformed to the Langmuir model (L or H type) whereas a linear (C type) adsorption isotherm was obtained at higher concentrations of ta
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