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Title: | 探討還原劑對於乳清蛋白類澱粉纖維形成之影響 Examining the Effect of Reducing Agent on the Amyloid Fibril Formation of Alpha-Lactalbumin |
Authors: | Pu Wang 王卜 |
Advisor: | 王勝仕(Sheng-Shih Wang) |
Keyword: | 類澱粉纖維,乳清蛋白,溶菌酶,穀胱甘肽,還原劑,雙硫鍵, amyloid,fibril,alpha-lactalbumin,lysozyme,glutathione,reducing agent,disulfide bond, |
Publication Year : | 2008 |
Degree: | 碩士 |
Abstract: | 到目前為止,已知至少二十種人類蛋白質能夠產生纖維狀結構的類澱粉聚集,同時造成類澱粉症。類澱粉症是由於蛋白質結構的摺疊錯誤造成聚集的生成,此類聚集以纖維狀的結構彼此糾結,並且導致細胞與組織的病變。雖然知道類澱粉纖維會誘發疾病,但是對其病理機制至今仍無法完全了解。
本研究利用生物體內常見的穀胱甘肽(glutathione, GSH)當作還原劑,透過不同濃度GSH的添加來觀察雞蛋白溶菌酶(hen-egg-white lysozyme)和alpha-乳清蛋白(alpha-lactalbumin)兩種蛋白質產生類澱粉纖維行為之影響,並針對了GSH與蛋白質中雙硫鍵的反應來進行探討。本研究利用了多種分析方法,結果發現在55 oC與55 rpm之環境下可以成功地將溶菌酶與alpha-乳清蛋白誘導產生類澱粉纖維;添加5 mM GSH可以完全抑制alpha-乳清蛋白形成類澱粉纖維,而需10 mM GSH才能完全抑制溶菌酶形成類澱粉纖維;隨著GSH濃度的上升可以明顯地增加溶菌酶類澱粉纖維成長之延遲期,但是對於alpha-乳清蛋白幾乎沒有影響。當alpha-乳清蛋白與溶菌酶放置於GSH環境中超過24小時後,電泳結果會出現裂解的現象。從DTNB-GdnHCl的實驗中發現,alpha-乳清蛋白中的雙硫鍵會隨著GSH的添加而造成斷裂,GSH濃度越高雙硫鍵斷裂數目越多,至於未添加GSH的alpha-乳清蛋白並沒有發現雙硫鍵的斷裂。然而,未添加GSH的溶菌酶就已經有雙硫鍵的斷裂,而添加不同濃度的GSH並不會使溶菌酶雙硫鍵斷裂數目增加,但卻會減緩雙硫鍵的斷裂速度。本研究之結果除了有助於解釋alpha-乳清蛋白與溶菌酶形成類澱粉纖維之機制,以及雙硫鍵的斷裂對於類澱粉纖維之影響外,並可更進一步地對類澱粉症之抑制提供有效的策略。 At least twenty different human proteins can form species with fibrillar structure resulting in a variety of human diseases called amyloidosis. These proteins are capable of misfolding or self-assembling into stable fibrils with a characteristic cross-beta pleated sheet secondary structure. Although amyloid diseases have been the center of intense research, the role of amyloid protein and the toxicity mechanisms that mediate its biological responses remain largely unknown. In this research, we examined the effects of reduced form of glutathione (GSH) on the in vitro fibrillogenesis of bovine alpha-lactalbumin and hen-egg-white lysozyme at acidic pH (pH 2.0) and also focused on the interaction(s) between GSH and the disulfide bonds of these two proteins. Using ThT fluorescence, Congo red binding, ANS-fluorescence, and circular dichroism spectroscopy, we found that the native structure of alpha-lactalbumin or lysozyme was successfully converted into amyloid fibrils at 55 oC and 55 rpm. The formation of fibrils was completely inhibited in the presence of 5 mM and 10 mM GSH for alpha-lactalbumin and lysozyme, respectively. The growth of lysozyme fibrils was significantly delayed by the increase in GSH concentration but this behavior was not observed in the case of alpha-lactalbumin. When alpha-lactalbumin or lysozyme incubated with GSH for more than 24 hours, the acid cleavage/fragmentation was observed on the SDS-PAGE gels. We showed in the results of sulfhydryl content determination the number of disrupted disulfide bonds of alpha-lactalbumin, starting from 0 in its native state, increased with increasing concentration of GSH. As for lysozyme, samples with or without GSH had broken disulfide bridges and the presence of GSH otherwise reduced the rate of disulfide bond disruption. We believe that the outcome from this work can not only help explain the mechanisms of the fibril formation for alpha-lactalbumin and lysozyme but also explore the roles of disulfide bond in amyloid fibrils. Furthermore, results from this study provide potential inhibitory strategies against the amyloid formation associated with amyloid diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37589 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 化學工程學系 |
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