奈米二氧化鈰及氧化鋅對溶菌酶結構及活性之影響

Effects of Nano-Cerium Oxide and Zinc Oxide on the Structure and Activity of Lysozyme

賴佳閔(Chia-Min Lai)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10978

摘要

奈米粒子雖因其優越的物化特性而被廣泛地應用於電子、化妝品、與生醫等領域，然而其使用是否會對人體造成危害，仍有待進一步之研究。本研究以奈米二氧化鈰與奈米氧化鋅為系統，利用紫外光-可見光分光光度計、螢光光譜儀、圓二色光譜儀、界面電位分析儀等儀器及分析方法探討上述奈米粒子於pH7.4，37℃下對母雞蛋白溶菌酶之結構與活性的影響。實驗結果顯示奈米二氧化鈰不論吸附量，Freundlich親和常數，吸附強度與結合位點數都比氧化鋅高。於蛋白質二、三級結構方面，溶菌酶吸附上奈米二氧化鈰會使α-helix比例明顯下降且β-sheet及unorder比例上升；且由自體螢光光譜，螢光淬滅實驗、與Nile Red分析可知，溶菌酶之疏水區域有裸露之現象。相較於溶菌酶吸附於氧化鋅則不改變其二級結構，其疏水區域也只有些微裸露。此外，吸附於二氧化鈰上之溶菌酶活性有下降趨勢；而氧化鋅則幾乎保持其活性。氧化鋅對溶菌酶之穩定性也可由化學誘導蛋白質開展分析得之。於蛋白質二聚體與單體間之比例可發現，添加奈米粒子皆有促進二聚體生成之現象。由上述實驗結果推測溶菌酶之結構與活性改變與其吸附型態有關；推論之所以有不同吸附型態之產生可能與溶菌酶表面濃度與粒子表面性質相關。吾人推測溶菌酶以活性區對面之正電區域與奈米粒子表面鍵結，而由於氧化鋅表面溶菌酶多而限制溶菌酶結構開展；但二氧化鈰表面溶菌酶濃度較小，且溶菌酶正電區附近之極性胺基酸可能與二氧化鈰表面以氫鍵鍵結，加上曲率較大故會對結構造成扭曲破壞，進而影響其活性。我們相信本論文之結果將有助於對奈米粒子與生物分子間交互作用之瞭解。

關鍵字

奈米粒子 ； 二氧化鈰 ； 氧化鋅 ； 溶菌酶結構 ； 活性 ； 鍵結方位

並列摘要

Nanoparticles are widely used in the fields of electronics, cosmetics, and biomedical given their superior physicochemical properties. However, nanoparticles may have adverse influence on the functions of proteins or impose potential threat to our lifes upon entering the human body.Using hen egg-white lysozyme as a model protein, we made attempts to examine the effects of two kinds of nanoparticles, nano-cerium oxide and nano-zinc oxide, on the structure and activity of hen egg white lysozyme in the condition of pH 7.4 and 37℃ using UV/vis spectrophotometer, fluorescence spectroscopy, circular dichroism spectroscopy, zetasizer , Nile red assay, and glutaraldehyde cross-linking with SDS-PAGE. Our experimental results demonstrate that the adsorption capacity, Freundlich affinity constant, adsorption strength and number of binding sites of lysozyme adsorption on nano-cerium oxide are larger than those on nano-zinc oxide. Also, the conformation of lysozyme upon its adsorption onto cerium oxide experiences transition of α-helix to β-sheet and un-order secondary structures. Intrinsic fluorescence spectroscopy, fluorescence quenching, and Nile red analyses show that the hydrophobic regions of lysozyme are exposed to solvent. For the adsorption of lysozyme onto nano-zinc oxide, almost no change is detected in the secondary structure, and the hydrophobic regions are only slightly exposed. In addition, the enzyme activity of lysozyme decreased when adsorbing onto the nano-cerium oxide, whereas the adsorption onto zinc oxide had shown no influence on its enzyme activity. Chemically induced protein unfolding analysis also pointed out that lysozyme adsorbed onto the nano-zinc oxide was more stable than that onto the cerium oxide . Furthermore, glutaraldehyde cross-linking studies indicated that a higher percentage of dimeric species of lysozyme were produced as compared to the monomeric species when lysozyme is conjugated with either the nanto-cerium oxide or nano-zinc oxide. Our results suggest that the changes of lysozyme structure and activity are correlated with its adsorption patterns, which may be associated with the surface concentration of lysozyme on nanoparticles and the surface properties of nanoparticles. We speculate that lysozyme adsorbs to the surface of nanoparticles using its positively charged patch on the protein surface, which is located at the opposite side of its active site. Moreover, the concentration of lysozyme adsorption on the nano-zinc oxide is significantly high so that the unfolding of lysozyme molecules is less likely to occur, thus no marked loss in enzyme activity is observed. On the contrary, the concentration of lysozyme adsorption on the nano-cerium oxide is relatively lower and some polar amino acid near the lysozyme’s positively charged patch may interact with the surface of the nano-cerium oxide through hydrogen bonding. This strong interaction/adsorption would lead to the distortion and destruction of lysozyme structure, thereby reducing its emzyme activity. We believe the outcome form this thesis may contribute to a better understanding of the interactions between nanoparticles and biomolecules.

並列關鍵字

nanoparticle ； cerium oxide ； zinc oxide ； Hen egg white lysozyme(HEWL) ； structure ； activity ； binding site

參考文獻

參考文獻

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