氧化還原緩衝液組成對於母雞蛋白類澱粉纖維形成之影響

目前研究發現，人體中有超過二十種以上的蛋白質會因為摺疊錯誤而產生類澱粉纖維沉積，更嚴重者，甚至會造成致命性的疾病產生，而這些疾病被統稱為類澱粉症。在類澱粉症發病的過程中，蛋白質會自我聚集成富含beta-sheet結構的類澱粉纖維。雖然目前已有許多針對類澱粉纖維生成的研究結果發表，但是對於蛋白質分子結構變化的詳細機制卻仍有待釐清。本研究利用不同的半胱胺酸及胱胺酸氧化還原組成比例，來探討氧化還原緩衝液組成對於母雞蛋白溶菌酶形成類澱粉纖維之影響。實驗中，藉由數種光學測量方法及穿透式電子顯微鏡的輔助，吾人可發現氧化還原緩衝液中的半胱胺酸可有效抑制母雞蛋白類澱粉纖維之生成。在添加有1 mM半胱胺酸的組別中，母雞蛋白類澱粉纖維的形成可受到約30 %的抑制，而添加2 mM半胱胺酸之組別，則可達到約70 %之抑制效果，因此發現半胱胺酸的抑制效果與其濃度有關，當加入之半胱胺酸濃度越高，則抑制類澱粉纖維生成之效果越佳。相對於半胱胺酸與濃度成正比的抑制效果，其氧化態之胱胺酸則僅能使類澱粉纖維生長過程中之延遲期延長，並減緩類澱粉纖維生長之速率，但對於最終生成類澱粉纖維的總量則無明顯影響。此外，從本實驗的結果也發現，半胱胺酸的存在將使母雞蛋白溶菌酶內的雙硫鍵破壞程度增加，添加1 mM的半胱胺酸可使蛋白質內約53 %的雙硫鍵斷裂，而添加2 mM的半胱胺酸則可使蛋白質內雙硫鍵的斷裂程度提升至約79 %，這樣的趨勢與半胱胺酸抑制類澱粉纖維生成之效果類似，因此半胱胺酸抑制類澱粉纖維之形成應該與蛋白質內雙硫鍵之斷裂有關。藉由本實驗的結果證實，當加入半胱胺酸時，蛋白質內的雙硫鍵會被破壞並達到抑制類澱粉纖維生成之效果。且由於添加2 mM半胱胺酸時，蛋白質內雙硫鍵的斷裂程度達到79 %，但其抑制類澱粉纖維生成之效果卻僅為70 %，因此在文獻的輔助下，吾人推測和母雞蛋白溶菌酶形成類澱粉纖維相關之Cys64-Cys80應該是最後一對被打斷的雙硫鍵，當此對雙硫鍵被破壞，母雞蛋白溶菌酶將不再生成類澱粉纖維。藉由本研究的結果，或許可以幫助吾人釐清在類澱粉纖維生成的過程中，具雙硫鍵的蛋白質分子結構變化及詳細機制，甚至在疾病的預防及治療上有所貢獻。

關鍵字

溶菌酶類澱粉纖維；氧化還原對；半胱胺酸；胱胺酸；雙硫鍵

並列摘要

As of now, more than twenty different human proteins can fold abnormally resulting in the formation of amyloid fibril deposits and several lethal degenerative diseases, which are called amyloidoses or amyloid diseases. During the progression of the diseases, these proteins can self-assemble into stable fibrils with extensive beta-sheet conformation. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current study is aimed at exploring the effect of varying ratio of cysteine and cystine on the fibrillogenesis of hen egg-white lysozyme. Via numerous spectroscopic techniques and transmission electron microscopy, our data revealed that the inhibition of lysozyme amyloid formation by cysteine in the redox buffer followed a concentration-dependent fashion. For instance, approximately 30 % or 70% inhibition of fibrillogenesis was observed in the presence of 1 mM or 2 mM cysteine, respectively. In contrast, the oxidized form of cysteine, cystine, nevertheless, did not influence the final level of fibrillogenesis although it lengthened the lag period and decreased the growth rate of fibril formation. Moreover, we observed in our study that the presence of cysteine led to a higher degree of disulfide bond disruption. About 53 % and 79% of disulfide bond disruptions were detected when 1 mM and 2 mM cysteine were added, respectively. It could be concluded from our results that there existed a connection between the inhibition level of fibrillogenesis and the percentage of native disulfide bond disruption for lysozyme. Our results demonstrated that the disulfide bonds could be disrupted by the addition of cysteine and thus leading to significant inhibitory effects against fibril formation. According to the results from this study and the literature, we suggested that the disulfide bridge Cys64-Cys80 which is strongly associated with lysozyme fibrillogenesis would be the last broken disulfide bond. Therefore, the fibril formation would be suppressed when this disulfide bond was broken. The outcome from this work may aid in comprehending the molecular mechanism(s) of fibrillogenesis for disulfide bond-containing amyloid proteins and development of effective therapeutics for amyloidogenic diseases.

並列關鍵字

lysozyme ； amyloid fibril ； redox pair ； cysteine ； cystine ； disulfide bond

參考文獻

120. 周上偉. 氧化還原對之於類澱粉蛋白質聚集行為的影響. 台灣大學化學工程學研究所 (2006).

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氧化還原緩衝液組成對於母雞蛋白類澱粉纖維形成之影響

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