以光譜法探討棒麴毒素與人血清白蛋白之相互作用

Spectroscopic Study on the Interaction Between Patulin and Human Serum Albumin

黃文彥(Wen-Yan Huang)

指導教授 ： 錢偉鈞

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2018年)

摘要

棒麴毒素為普遍存在於蘋果與其加工製品中的黴菌毒素，攝取過多可造成腸道出血、呼吸困難、腎臟損傷、致畸性和致癌作用等急、慢性症狀，人血清白蛋白是血漿中含量最豐富的蛋白質，有良好的配體結合能力，為許多內源性和外源性化合物之載體。本文透過儀器分析探討棒麴毒素及人血清白蛋白之相互作用，實驗結果發現，棒麴毒素會與人血清白蛋白產生鍵結形成複合物，引發人血清白蛋白之二級結構改變而發生靜態淬熄且伴隨動態淬熄。UV/Vis光譜儀結果可觀察到人血清白蛋白訊號峰之吸收值隨著棒麴毒素濃度增加而增加並產生紅位移，螢光檢測在溫度288K、298K、310K下，棒麴毒素與人血清白蛋白之結合常數分別為3.22×10^7 M^-1、4.51×10^6 M^-1、8.06×10^5 M^-1，兩分子之間存在一個結合點，透過其反應焓∆H^0及熵∆S^0確認鍵結方式是以氫鍵與分散力為主要驅動力，而三種溫度下之自由能∆G^0分別為-41.20 kJ/mol、-38.31 kJ/mol、-34.84 kJ/mol，表示其為自發性反應，圓二色光譜結果發現棒麴毒素會改變人血清白蛋白之二級結構，造成其α-helix結構含量增加，也因與蛋白質大分子之間的氫鍵關係使得棒麴毒素在水中之擴散速率降低，核磁共振光譜儀中的DOSY實驗結果測得棒麴毒素與人血清白蛋白之結合常數為2.279×10^3 M^-1，而自由能∆G^0為-19.16 kJ/mol，表示兩分子確實形成複合物且其鍵結過程為自發性反應。

關鍵字

棒麴毒素 ； 人血清白蛋白 ； 複合物 ； 螢光淬熄 ； 核磁共振光譜儀

並列摘要

Patulin (PAT) is a mycotoxin commonly found in apple and its processed products, when excessively intake, may cause acute and chronic symptoms such as intestinal bleeding, respiratory difficulties, kidney damage, teratogenicity, and carcinogenesis. Human serum albumin (HSA) is the most abundant protein in plasma. With the ability to interact with a variety of ligands, HSA is a carrier for many endogenous and exogenous compounds. This study explores the interaction between PAT and HSA through instrumental analysis. The red shift of the HSA UV/Vis spectra and a static quench in HSA fluorescence intensity with the introduction of PAT are the spectroscopic evidence for the interaction between PAT and HSA. Analysis of fluorescence experimental results showed that, at the temperature of 288K, 298K, and 310K, the binding constants of PAT to HSA was 3.22×10^7 M^-1, 4.51×10^6 M^-1, and 8.06×10^5 M^-1, respectively. Hydrogen bonding and Dispersing force are the main driving force for the formation of toxin-protein complex. The free energy at three temperatures are -41.20 kJ/mol, -38.31 kJ/mol, and -34.84 kJ/mol, respectively, indicating that it is a spontaneous reaction. Circular dichroism study showed that PAT would change the secondary structure of HSA during the formation of toxin-HSA complex and could induce stability of α-helix structure in HSA. These results are confirmed by NMR DOSY experiments. The binding constant of PAT to HSA was measured as 2.279×10^3 M^-1, and the derived free energy was -19.16 kJ/mol. Formation of PAT-HSA complex is a spontaneous process.

並列關鍵字

Patulin ； Human serum albumin ； Complex ； Fluorescence quenching ； NMR

參考文獻

1.Krska, R., “Mycotoxins,” Analytical and Bioanalytical Chemistry, Vol. 395, No. 5, pp. 1203 (2009).

註：黴菌毒素對人體之危害

2.Zöllner, P., Mayer-Helm, B., “Trace Mycotoxin Analysis in Complex Biological and Food Matrices by Liquid Chromatography-Atmospheric Pre-ssure Ionisation Mass Spectrometry,” Journal of Chromatography A, Vol. 1136, pp. 123-69 (2007).

註：目前常見之黴菌毒素分析方法

3.Hussein, H. S., Brasel, J. M., “Toxicity, Metabolism, and Impact of Myco-toxins on Humans and Animals,” Toxicology, Vol. 167, No. 2, pp. 101-134 (2001).