本文選題：次血紅素 + 電子順磁共振　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：模擬酶是通過人工合成出的一類具有天然酶生物特性的催化劑。模擬酶是一種仿生高分子,通過模擬天然酶的活性中心和作用機(jī)理使其具備天然酶的一些催化特性。天然酶是從生物體內(nèi)提取出來的一類具備催化特性的蛋白質(zhì)。天然酶有很多優(yōu)點(diǎn),比如專一催化一類化學(xué)或者生物反應(yīng)、具有很高的催化效率等。但是天然酶作為蛋白質(zhì),在體外進(jìn)行催化反應(yīng)時(shí)很容易受物理因素或者化學(xué)因素的影響,因此在工業(yè)生產(chǎn)中天然酶往往并不能取代化學(xué)催化劑。模擬酶具有簡(jiǎn)單的催化結(jié)構(gòu),可以解決天然酶不穩(wěn)定的缺點(diǎn)。20世紀(jì)60年代至今,多種多樣的模擬酶被人工合成出來,并且應(yīng)用于工業(yè)生產(chǎn)和環(huán)境化學(xué)中。通過研究模擬酶的結(jié)構(gòu)、活性中心、與底物作用的方式等,可以更好的改進(jìn)模擬酶的催化性能。次血紅素(Deuterohemin,Dh)是通過人工合成的一種鐵卟啉化合物。次血紅素區(qū)別于天然的血紅素,缺少了兩個(gè)乙烯基,由于乙烯基易被氧化,因此這樣的結(jié)構(gòu)特征可以使次血紅素在氧化底物過程中更加穩(wěn)定。在1971年,就有研究表明次血紅素可以在過氧化氫的存在下氧化有機(jī)底物,雖然活性不高,但是這個(gè)結(jié)果說明了次血紅素可以模擬過氧化物酶活性,也為研究次血紅素化合物的功能奠定了基礎(chǔ)。金屬卟啉模擬過氧化物酶活力取決于中心金屬的電子結(jié)構(gòu)以及額外配體與金屬卟啉環(huán)之間的相互作用。次血紅素雖然存在金屬中心,可以通過金屬的變價(jià)模擬過氧化物酶活性,但是其缺少了一個(gè)額外的配體,導(dǎo)致了很低的過氧化物酶活性。根據(jù)微過氧化物酶(Microperoxidases,MPs)的側(cè)鏈設(shè)計(jì)出了一個(gè)含有六個(gè)氨基酸的肽段(Ala-His-Thr-Val-Glu-Lys),并將這個(gè)肽段共價(jià)連接在了次血紅素的羧基上。我們將這個(gè)人工合成的次血紅素肽,命名為次血紅素六肽(Dh HP-6)。因?yàn)榻说慕M氨酸可以與活性中心鐵進(jìn)行配位,因此其活性高于沒有肽段的次血紅素。為了進(jìn)一步研究組氨酸對(duì)次血紅素活性的影響,我們用丙氨酸代替了組氨酸,合成了一個(gè)新的肽段(Ala-Ala-Thr-Val-Glu-Lys),并將這個(gè)肽段連接在了次血紅素的羧基上(Dh AP-6)。利用紫外可見吸收光譜,我們對(duì)兩種次血紅素肽進(jìn)行了結(jié)構(gòu)的分析,并且進(jìn)行了酶活性的測(cè)定。我們發(fā)現(xiàn),Dh AP-6的過氧化物酶活性低于Dh HP-6。因此,組氨酸的配位,對(duì)提高次血紅素的過氧化物酶活性起到了關(guān)鍵的作用。隨后,我們對(duì)Dh HP-6的過氧化物酶活性進(jìn)行了反應(yīng)條件的優(yōu)化,并在最適條件下,進(jìn)行了動(dòng)力學(xué)的研究,發(fā)現(xiàn)Dh HP-6相比于辣根過氧化物酶(HRP)具有很高的底物親和性。酶的催化反應(yīng)是需要水作為介質(zhì)才能進(jìn)行的,而有機(jī)溶劑可以破壞天然酶中的氫鍵和剝奪酶分子表面的必需水,因此在有機(jī)介質(zhì)中天然酶的結(jié)構(gòu)很容易被破壞,導(dǎo)致活性中心的改變,從而使酶活力下降或者使酶失活。由于Dh HP-6結(jié)構(gòu)簡(jiǎn)單,不具備蛋白質(zhì)復(fù)雜的三維結(jié)構(gòu),所以在有機(jī)溶劑中也表現(xiàn)出了很好的酶活性。我們發(fā)現(xiàn)Dh HP-6的過氧化物酶活性與有機(jī)溶劑的極性有關(guān),溶劑的極性越大,Dh HP-6的過氧化物酶活性越高。在15%的甲醇溶液中,Dh HP-6的活性最高,是Horseradish Peroxidase(HRP)活性的15倍。為了可以更好的利用Dh HP-6的過氧化物模擬酶活性,通過Dh HP-6與Cu_3(PO_4)_2自組裝,將Dh HP-6固定化。發(fā)現(xiàn)Dh HP-6與Cu_3(PO_4)_2自組裝之后形成了一種類似于花的納米結(jié)構(gòu),我們稱之為Dh HP-6-Cu_3(PO_4)_2納米花。利用掃描電鏡、固體紫外、固體紅外等手段,對(duì)Dh HP-6-Cu_3(PO_4)_2納米花進(jìn)行了結(jié)構(gòu)的表征。通過電子順磁共振光譜,我們首次從實(shí)驗(yàn)角度解釋了納米花形成的機(jī)理。形成納米花之后,Dh HP-6對(duì)底物的親和性進(jìn)一步提高。在形成納米花的過程中,原本聚集的Dh HP-6,分散的固定到了Cu_3(PO_4)_2上,使得Dh HP-6的活性中心暴露,因此Dh HP-6-Cu_3(PO_4)_2納米花的酶活力和對(duì)底物的親和性都有所提高。我們嘗試將Dh HP-6-Cu_3(PO_4)_2納米花應(yīng)用于苯酚的檢測(cè),并發(fā)現(xiàn)其具有很好的LOD值和靈敏度。而且,多次使用和長(zhǎng)時(shí)間在緩沖溶液中貯存也沒有明顯的活力損失,因此Dh HP-6-Cu_3(PO_4)_2納米花是一種具有很好的應(yīng)用前景的生物催化劑。最后,我們利用理論計(jì)算,研究了Dh HP-6氧化苯酚的反應(yīng)機(jī)理。研究發(fā)現(xiàn),在氧化苯酚的過程中,以兩態(tài)反應(yīng)模式(TSR)進(jìn)行,且二重態(tài)和四重態(tài)的能量和結(jié)構(gòu)都很接近。電子結(jié)構(gòu)分析表明,O-H鍵的活化是一個(gè)PCET機(jī)制,電子由苯酚轉(zhuǎn)移到了卟啉環(huán)上,氫質(zhì)子轉(zhuǎn)移到了金屬氧上,而金屬中心的電子未發(fā)生變化。二重態(tài)和四重態(tài)的不同在于前者是α電子轉(zhuǎn)移,后者是β電子轉(zhuǎn)移。
[Abstract]:A mimic enzyme is a kind of synthetic catalyst with natural enzyme biological properties. The analogue enzyme is a bionic polymer by simulating the active center and mechanism of natural enzyme to make it have some catalytic properties of natural enzyme. Natural enzyme is a kind of protein with catalytic properties extracted from the organism. There are many advantages, such as catalyzing a class of chemical or biological reactions, and having high catalytic efficiency. But natural enzymes are easily affected by physical or chemical factors when they are used as proteins in vitro. Therefore, natural enzymes can not replace chemical catalysts in industrial production. The single catalytic structure can solve the disadvantage of natural enzyme instability..20 century since 60s, a variety of analog enzymes have been synthesized and applied to industrial production and environmental chemistry. By studying the structure of the enzyme, the activity center, the way of the action of the substrate and so on, the catalytic performance of the analogue enzyme can be improved better. Deuterohemin (Dh) is an artificially synthesized iron porphyrin compound. The heme is different from the natural heme and lacks two vinyl groups. Because the vinyl group is easily oxidized, this structural feature can make the heme more stable during the oxidation of the substrate. In 1971, a study showed that heme could be used. The oxidation of organic substrates in the presence of hydrogen peroxide is not highly active, but this result shows that heme can simulate peroxidase activity and also provide a basis for the study of the function of heme compounds. The interaction between porphyrin rings. Although heme exists in the metal center, it can simulate peroxidase activity through the metal variation, but it lacks an extra ligand, which leads to a very low peroxidase activity. A six amino acid is designed according to the side chain of the Microperoxidases (MPs). The peptide segment (Ala-His-Thr-Val-Glu-Lys), which is covalently linked to the carboxyl group of the heme, is named the heme heme six peptide (Dh HP-6), because the near terminal histidine can be coordinated with the active central iron, so its activity is higher than the heme without the peptide. One step was to study the effect of histidine on the activity of heme. We used alanine instead of histidine and synthesized a new peptide segment (Ala-Ala-Thr-Val-Glu-Lys), which was connected to the carboxyl group of heme (Dh AP-6). The structure of two heme peptides was analyzed by UV visible absorption spectrum. The activity of the enzyme was determined. We found that the peroxidase activity of Dh AP-6 was lower than that of Dh HP-6., so the coordination of histidine played a key role in improving the activity of the peroxidase in the heme. Then, we optimized the activity of the peroxidase in Dh HP-6 and carried out the power under the optimum conditions. Studies have found that Dh HP-6 has a very high substrate affinity compared to horseradish peroxidase (HRP). The catalytic reaction of the enzyme is required by water as a medium, and organic solvents can destroy the hydrogen bonds in natural enzymes and the necessary water on the surface of the enzyme molecules. Therefore, the structure of natural enzymes in organic medium is easily destroyed. The changes in the active center make the activity of the enzyme declines or inactivate the enzyme. Because Dh HP-6 has a simple structure and does not have a complex three-dimensional structure of protein, it also shows good enzyme activity in the organic solvent. We found that the peroxidase activity of Dh HP-6 is related to the polarity of organic solvents, the greater the polarity of the solvent, the Dh HP-6 The higher the activity of peroxidase is, the activity of Dh HP-6 is the highest in 15% methanol solution, which is 15 times of the activity of Horseradish Peroxidase (HRP). In order to make better use of the activity of the peroxidase in Dh HP-6, it will be fixed by Dh HP-6 and Cu_3 (PO_4). The nanostructures, which are similar to flowers, are called Dh HP-6-Cu_3 (PO_4) _2 nanoscale flowers. The structure of Dh HP-6-Cu_3 (PO_4) _2 nanoscale was characterized by scanning electron microscopy, solid ultraviolet and solid infrared, and the mechanism of nanoscale formation was explained by the electron paramagnetic resonance spectrum. Then, the affinity of Dh HP-6 to the substrate was further improved. In the process of forming the nanoscale, the original aggregation of Dh HP-6 was fixed to Cu_3 (PO_4) _2, making the active center of Dh HP-6 exposed, so the enzyme activity of Dh HP-6-Cu_3 (PO_4) and the affinity to the substrate were improved. It was applied to the detection of phenol and found that it had good LOD value and sensitivity. Moreover, the storage of Dh HP-6-Cu_3 (PO_4) _2 nanoscale was a kind of biocatalyst with good application prospect. Finally, we used theoretical calculation to study Dh HP. In the process of oxidation of phenol by -6, it is found that in the process of oxidation of phenol, the two state reaction mode (TSR) is carried out, and the energy and structure of the double and four states are close. The electronic structure analysis shows that the activation of the O-H bond is a PCET mechanism, the electron transfer from phenol to the porphyrin ring and the hydrogen proton transfer to the metal oxygen, and gold. The electron in the center is not changed. The difference between the duality and the four heavy States is that the former is alpha electron transfer and the latter is beta electron transfer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：O643.36

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