SPR生物传感膜的构筑及其对氨基酸的手性识别研究
发布时间：2019-03-03 11:42
【摘要】：表面等离子体共振(SPR)技术作为一种新型灵敏度极高的免标记、高通量、低耗量、无损检测生物传感技术,是当前研究分子相互作用最重要的方法之一,可以实时动态研究各种分子相互作用全过程,在生命科学领域和药物研究与开发等方面有非常独特的优势。 由于氨基酸、多糖、蛋白质、DNA等生物大分子多数都具有手性结构,使得生命过程中产生了众多复杂的手性识别现象,手性物质的立体选择性导致手性分子与生命体内源性大分子相互作用存在显著差异。因此开展手性识别研究对生物、医药、化学领域都有非常重要的意义。 本论文利用SPR技术,以牛血清白蛋白(BSA)和人血清白蛋白(HSA)为探针构筑了手性识别传感膜,研究了这两种生物蛋白与不同种类的氨基酸对映异构体的相互作用,通过考察相互作用过程中结合速率、解离速率和亲和力大小等差异进行手性识别研究,并对结合特异性及手性识别机理进行了探索。 第一,在25℃,pH7.4的生理条件下对L-和D-色氨酸进行了手性识别的动力学研究,考察了pH、蛋白浓度和离子强度等标记条件对蛋白标记量的影响,获得了BSA、HSA与色氨酸对映异构体相互作用的动力学参数,并对结合机理进行了探讨。 第二,通过测定不同pH、离子强度、温度对BSA和HSA与L-和D-色氨酸结合的亲和力影响,对两种蛋白与色氨酸对映异构体结合的特异性进行了研究。 第三,研究了BSA、HSA与苯丙氨酸、精氨酸、甲硫氨酸以及组氨酸4种氨基酸的8种不同对映体结合的动力学相关作用过程,并进行了手性识别研究。 实验结果显示,两种血清白蛋白在与每种氨基酸分子的L-和D-型异构体相互作用过程中都存在明显的动力学差异,且两种蛋白与每种氨基酸L型异构体的亲和力均大于D型。不同pH、离子强度、温度对BSA、HSA与L-和D-色氨酸结合的亲和力存在影响,但结果都显示L-型变化程度明显大于D-型,表明BSA、HSA与L-色氨酸结合存在特异性结合位点；热力学数据分析表明疏水作用在色氨酸对映体与血清白蛋白的结合中起主导作用,但静电作用对结合也有一定的贡献,而血清白蛋白与D-色氨酸的结合只是非特异性吸附；实验还表明,响应值大小并不会影响到结合亲和力,这和小分子自身质量、结合特异性等有关。 本论文通过实时监测血清白蛋白与手性氨基酸的结合过程,获得了可靠的结合动力学数据和亲和力信息,这为生命体特异性选择吸收手性氨基酸和手性药物提供了重要的量论依据,对手性识别与新药研发具有一定的指导意义。
[Abstract]:Surface plasmon resonance (SPR) (SPR) is one of the most important methods to study molecular interaction, which is a new label-free technique with high sensitivity, high throughput, low consumption and nondestructive detection. It can study the whole process of molecular interaction in real-time and dynamically. It has unique advantages in the field of life sciences and drug research and development. Most of the biological macromolecules, such as amino acids, polysaccharides, proteins, DNA and so on, have chiral structures, which lead to many complex chiral recognition phenomena in the process of life. The stereoselectivity of chiral substances leads to significant differences in the interaction between chiral molecules and endogenous macromolecules in life. Therefore, it is very important to carry out chiral recognition research in biology, medicine and chemistry. In this paper, the chiral recognition sensing membranes were constructed using bovine serum albumin (BSA) and human serum albumin (HSA) probes by SPR technique, and the interaction between the two proteins and different amino acid enantiomers was studied. Chiral recognition was studied by investigating the differences of binding rate, dissociation rate and affinity in the interaction process, and the binding specificity and chiral recognition mechanism were explored. Firstly, the kinetics of chiral recognition of L-and D-tryptophan was studied under the physiological condition of pH7.4 at 25 鈩，
本文编号：2433665
[Abstract]:Surface plasmon resonance (SPR) (SPR) is one of the most important methods to study molecular interaction, which is a new label-free technique with high sensitivity, high throughput, low consumption and nondestructive detection. It can study the whole process of molecular interaction in real-time and dynamically. It has unique advantages in the field of life sciences and drug research and development. Most of the biological macromolecules, such as amino acids, polysaccharides, proteins, DNA and so on, have chiral structures, which lead to many complex chiral recognition phenomena in the process of life. The stereoselectivity of chiral substances leads to significant differences in the interaction between chiral molecules and endogenous macromolecules in life. Therefore, it is very important to carry out chiral recognition research in biology, medicine and chemistry. In this paper, the chiral recognition sensing membranes were constructed using bovine serum albumin (BSA) and human serum albumin (HSA) probes by SPR technique, and the interaction between the two proteins and different amino acid enantiomers was studied. Chiral recognition was studied by investigating the differences of binding rate, dissociation rate and affinity in the interaction process, and the binding specificity and chiral recognition mechanism were explored. Firstly, the kinetics of chiral recognition of L-and D-tryptophan was studied under the physiological condition of pH7.4 at 25 鈩，
本文编号：2433665
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