本文選題：結(jié)合位點(diǎn)　切入點(diǎn)：人血清白蛋白　出處：《中國(guó)科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：人血清白蛋白(Human serum albumin)是人體內(nèi)循環(huán)系統(tǒng)中含量最高的蛋白質(zhì),約占血清中蛋白含量的60%,通常健康成年人的血清中白蛋白的濃度約為50mg/mL(-600μM)。血清白蛋白的主要作用是結(jié)合并傳遞各種藥物以及內(nèi)源性代謝物至人體的各個(gè)器官組織。白蛋白體積較大,結(jié)構(gòu)復(fù)雜,含有585個(gè)氨基酸殘基,相對(duì)質(zhì)量為66 kDa,其單體由三個(gè)結(jié)構(gòu)域組成,其中每個(gè)結(jié)構(gòu)域又含有兩個(gè)子結(jié)構(gòu)域。因此,在白蛋白的表面會(huì)形成多個(gè)結(jié)合位點(diǎn),如脂肪酸結(jié)合位點(diǎn),金屬離子結(jié)合位點(diǎn)等。其中有兩個(gè)主要的藥物結(jié)合位點(diǎn),稱之為Sudlow's site Ⅰ和Sudlow's site Ⅱ。而藥物在體內(nèi)的藥效藥性,以及在血液中的有效濃度等,都與白蛋白上的這兩個(gè)結(jié)合位點(diǎn)密切相關(guān)。白蛋白上還有四個(gè)主要的金屬離子結(jié)合位點(diǎn),其中Site A為鋅離子的首要結(jié)合位點(diǎn)。而血清中的鋅離子大部分都結(jié)合在白蛋白上,因此對(duì)鋅離子結(jié)合位點(diǎn)的研究具有很高的重要性。晶體衍射是目前蛋白質(zhì)研究中主要的研究方法。然而,結(jié)晶過(guò)程以及樣品的配制需要消耗大量的時(shí)間,且繁瑣復(fù)雜。核磁共振技術(shù)作為一種新興的無(wú)損技術(shù)在蛋白質(zhì)的分析領(lǐng)域得到了廣泛的應(yīng)用。核磁共振技術(shù)最大的優(yōu)勢(shì)在于方便快捷,我們無(wú)需再對(duì)樣品進(jìn)行分離預(yù)處理,.也無(wú)需破壞樣品的完整性,從而大大降低了分離樣品所帶來(lái)的耗時(shí)和誤差。由于白蛋白體積過(guò)于龐大,氨基酸之間互相重疊包裹,因此普通的一維氫譜不僅譜峰變寬嚴(yán)重,而且譜峰之間相互重疊,大大的影響了我們對(duì)譜圖的分析。而常規(guī)的橫向弛豫濾波技術(shù)雖然可以濾掉寬包信號(hào),但同時(shí)也抑制掉了一些重要的信息。本課題中,我們通過(guò)引進(jìn)新的譜編輯技術(shù),可直接觀測(cè)天然豐度白蛋白的相互作用信息。(1) 我們首先建立了基于輻射阻尼效應(yīng)的WaterLOGSY譜編輯技術(shù)(RD-WaterLOGSY),通過(guò)該技術(shù)增強(qiáng)白蛋白信號(hào),然后通過(guò)引入橫向弛豫加權(quán)(T2W),濾掉白蛋白譜峰上的一些弛豫時(shí)間較短的寬包信號(hào),保留白蛋白表面與水分子具有快速交換或者強(qiáng)NOE的信號(hào)。結(jié)合常規(guī)二維TOCSY譜,我們發(fā)現(xiàn)該方法所觀察到的血清白蛋白信號(hào)主要來(lái)自于白蛋白表面的組氨酸。(2) 基于以上所建立的T2W-RD-WaterLOGSY技術(shù),我們可觀察離子與白蛋白的相互作用。我們發(fā)現(xiàn)在改變?nèi)芤旱膒H值時(shí),T2W-RD-WaterLOGSY技術(shù)所觀測(cè)的譜圖上低場(chǎng)區(qū)某些信號(hào)化學(xué)位移會(huì)發(fā)生明顯的移動(dòng),據(jù)此我們發(fā)現(xiàn)可以利用化學(xué)位移的相對(duì)變化來(lái)判斷溶液pH的變化。另外,當(dāng)向溶液中逐步加入金屬鋅離子時(shí),T2W-RD-WaterLOGSY譜圖上某些峰強(qiáng)度的變化與鋅離子濃度的變化成線性關(guān)系,表明T2W-RD-WaterLOGSY技術(shù)可以對(duì)鋅離子的結(jié)合起到指示的作用。(3) 基于以上T2W-RD-WaterLOGSY技術(shù),我們可以還可以快速觀察藥物與白蛋白的相互作用,判斷作用位點(diǎn)信息。我們向白蛋白溶液中加入了兩種不同的藥物,觀察隨著藥物濃度的增加,白蛋白信號(hào)的變化情況。發(fā)現(xiàn)該技術(shù)所觀測(cè)到的低場(chǎng)區(qū)的某些信號(hào)的化學(xué)位移和強(qiáng)度與特定藥物結(jié)合位點(diǎn)有關(guān)：當(dāng)藥物結(jié)合在Sudlow's site Ⅰ時(shí),這些低場(chǎng)區(qū)信號(hào)的強(qiáng)度發(fā)生改變,而當(dāng)藥物結(jié)合在Sudlow's site Ⅱ時(shí),這些信號(hào)并沒(méi)有發(fā)生明顯變化。(4) 我們利用T2W-RD-WaterLOGSY技術(shù)對(duì)血清中白蛋白進(jìn)行了檢測(cè),發(fā)現(xiàn)該技術(shù)同樣可以選擇性檢測(cè)血清中的白蛋白,消除其他小分子信號(hào)的影響。
[Abstract]:Human serum albumin (Human serum albumin) is a protein content in human body in the circulatory system of the highest, accounting for about 60% of the protein content in serum, serum albumin concentration generally healthy adults in approximately 50mg/mL (-600 M). The main function of serum albumin is a combination of various drugs and endogenous metabolites and transfer to human bodies organ albumin. Large volume, complex structure, containing 585 amino acid residues, the relative quality of 66 kDa, the monomer consisting of three domains, each domain contains two sub domains. Because of this, the formation of multiple binding sites on albumin surface, such as fatty acid binding sites. The metal ion binding sites. There are two main drug binding site, called the Sudlow's site I and Sudlow's II and site. In vivo efficacy of drug resistance, and the effective concentration in the blood Etc., are closely related with these two albumin binding sites. There are four main metal ion binding sites on albumin, which Site A is the primary binding sites of zinc ions and zinc ions in serum. Most with albumin, so it has a high importance of a point of zinc ion. Crystal diffraction is the main research method of the protein research. However, the crystallization process and the sample preparation need to consume a large amount of time, cumbersome and complex. The nuclear magnetic resonance technology as a new nondestructive technique has been widely applied in the field of protein analysis. The advantage of NMR technology is the most convenient. We need no separation of the samples pretreatment. There is no need to destroy the integrity of the sample, thereby greatly reducing the sample separation caused by the time-consuming and error. Due to the volume of albumin The amino acid is too large, overlapping parcels, so the ordinary one-dimensional hydrogen peak broadening spectrum not only seriously, and spectral overlapping peaks, greatly affected our analysis of the spectrum. Although conventional T2 filtering can filter out Henan wide packet signal, but also suppress some important the information. In this paper, we introduce a new spectral editing technology, interaction information direct observation of natural abundance albumin. (1) we first established the radiation damping effect of WaterLOGSY spectral editing technology based on (RD-WaterLOGSY), through the enhancement of albumin signal, and then through the introduction of transverse relaxation weighted (T2W), to filter out some of the albumin peak relaxation time shorter width packet signal, signal reservation albumin surface and water molecule has rapid exchange or strong NOE. Combined with the conventional 2D TOCSY spectrum, we found that Serum albumin is the signal observed mainly from albumin surface histidine. (2) set up above based on T2W-RD-WaterLOGSY technology, we can observe the interaction between ions and albumin. We found that the change in the pH value of the solution, T2W-RD-WaterLOGSY technology, the observed spectra on the low field region some chemical shift signal will obviously move, we found that the relative change can be used to determine the chemical shift variation of the solution of pH. In addition, when the solution to gradually add zinc ions, changes of T2W-RD-WaterLOGSY spectrum changes some peak intensity on the map and the linear relationship between the concentration of zinc ions, showed that combination of T2W-RD-WaterLOGSY technology for zinc ion pointing to the role. (3) the above interaction based on T2W-RD-WaterLOGSY technology, we can also observe fast drug and albumin, sentenced Off site information. We add two different drugs to albumin solution, observed with the increase of drug concentration, the changes of albumin signal. This technology observed in the low field signal region of some of the chemical shift and intensity and specific drug binding sites for drug binding in Sudlow's site: when I was the low field region, the intensity of the signal changes, and when the drug combination in Sudlow's site II, these signals were not changed obviously. (4) we use T2W-RD-WaterLOGSY technology to detect the serum albumin, this technology can also be selectively detected in serum albumin, the elimination of other small molecule signal.

【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李曉晶,馮江華,景鳳英,李欣宇,裴奉奎;Gd(DTPA-BIN)的熱力學(xué)性質(zhì)及與牛血清白蛋白作用的NMR研究[J];波譜學(xué)雜志;1999年06期

，

本文編號(hào)：1652274