發(fā)布時(shí)間：2019-03-03 11:42

【摘要】：表面等離子體共振(SPR)技術(shù)作為一種新型靈敏度極高的免標(biāo)記、高通量、低耗量、無損檢測(cè)生物傳感技術(shù),是當(dāng)前研究分子相互作用最重要的方法之一,可以實(shí)時(shí)動(dòng)態(tài)研究各種分子相互作用全過程,在生命科學(xué)領(lǐng)域和藥物研究與開發(fā)等方面有非常獨(dú)特的優(yōu)勢(shì)。 由于氨基酸、多糖、蛋白質(zhì)、DNA等生物大分子多數(shù)都具有手性結(jié)構(gòu),使得生命過程中產(chǎn)生了眾多復(fù)雜的手性識(shí)別現(xiàn)象,手性物質(zhì)的立體選擇性導(dǎo)致手性分子與生命體內(nèi)源性大分子相互作用存在顯著差異。因此開展手性識(shí)別研究對(duì)生物、醫(yī)藥、化學(xué)領(lǐng)域都有非常重要的意義。 本論文利用SPR技術(shù),以牛血清白蛋白(BSA)和人血清白蛋白(HSA)為探針構(gòu)筑了手性識(shí)別傳感膜,研究了這兩種生物蛋白與不同種類的氨基酸對(duì)映異構(gòu)體的相互作用,通過考察相互作用過程中結(jié)合速率、解離速率和親和力大小等差異進(jìn)行手性識(shí)別研究,并對(duì)結(jié)合特異性及手性識(shí)別機(jī)理進(jìn)行了探索。 第一,在25℃,pH7.4的生理?xiàng)l件下對(duì)L-和D-色氨酸進(jìn)行了手性識(shí)別的動(dòng)力學(xué)研究,考察了pH、蛋白濃度和離子強(qiáng)度等標(biāo)記條件對(duì)蛋白標(biāo)記量的影響,獲得了BSA、HSA與色氨酸對(duì)映異構(gòu)體相互作用的動(dòng)力學(xué)參數(shù),并對(duì)結(jié)合機(jī)理進(jìn)行了探討。 第二,通過測(cè)定不同pH、離子強(qiáng)度、溫度對(duì)BSA和HSA與L-和D-色氨酸結(jié)合的親和力影響,對(duì)兩種蛋白與色氨酸對(duì)映異構(gòu)體結(jié)合的特異性進(jìn)行了研究。 第三,研究了BSA、HSA與苯丙氨酸、精氨酸、甲硫氨酸以及組氨酸4種氨基酸的8種不同對(duì)映體結(jié)合的動(dòng)力學(xué)相關(guān)作用過程,并進(jìn)行了手性識(shí)別研究。 實(shí)驗(yàn)結(jié)果顯示,兩種血清白蛋白在與每種氨基酸分子的L-和D-型異構(gòu)體相互作用過程中都存在明顯的動(dòng)力學(xué)差異,且兩種蛋白與每種氨基酸L型異構(gòu)體的親和力均大于D型。不同pH、離子強(qiáng)度、溫度對(duì)BSA、HSA與L-和D-色氨酸結(jié)合的親和力存在影響,但結(jié)果都顯示L-型變化程度明顯大于D-型,表明BSA、HSA與L-色氨酸結(jié)合存在特異性結(jié)合位點(diǎn)；熱力學(xué)數(shù)據(jù)分析表明疏水作用在色氨酸對(duì)映體與血清白蛋白的結(jié)合中起主導(dǎo)作用,但靜電作用對(duì)結(jié)合也有一定的貢獻(xiàn),而血清白蛋白與D-色氨酸的結(jié)合只是非特異性吸附；實(shí)驗(yàn)還表明,響應(yīng)值大小并不會(huì)影響到結(jié)合親和力,這和小分子自身質(zhì)量、結(jié)合特異性等有關(guān)。 本論文通過實(shí)時(shí)監(jiān)測(cè)血清白蛋白與手性氨基酸的結(jié)合過程,獲得了可靠的結(jié)合動(dòng)力學(xué)數(shù)據(jù)和親和力信息,這為生命體特異性選擇吸收手性氨基酸和手性藥物提供了重要的量論依據(jù),對(duì)手性識(shí)別與新藥研發(fā)具有一定的指導(dǎo)意義。
[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，

本文編號(hào)：2433665