二硫鍵的氧化還原狀態對Trx融合赤霉酸誘導富含半胱氨酸蛋白內源熒光及變性過程影響

摘要：在采用親和層析、SDS-聚丙烯酰胺凝膠電泳(SDS-PAGE)對原核表達的赤霉酸誘導的富含半胱氨酸蛋白(Trx-GcGASA)進行純化、鑒定的基礎上,運用穩態熒光光譜手段研究了二硫蘇糖醇(DTT)、氧化型谷胱甘肽(GSSG)、過氧化氫、鹽酸胍(GdnHCl)對Trx-GcGASA內源熒光及變性過程的影響,發現(1)在中性緩沖體系中融合蛋白的內源熒光以305 nm的酪氨酸的熒光發射為主;(2)伴隨著二硫鍵還原,融合蛋白中色氨酸和酪氨酸的相對熒光強度比值從0.7變化至1.8倍左右;(3)經過0.5 mmol·L~(-1) GSSG、5 mmol·L~(-1)過氧化氫處理后,酪氨酸和色氨酸的熒光強度下降約12～21%;(4)無論是否采用1 mmol·L~(-1) DTT處理,6 mol·L~(-1)鹽酸胍均不能誘導融合蛋白徹底變性;(5)二硫鍵的存在與否影響了鹽酸胍誘導的變性過程.通過兩態模型擬合獲得Trx-GcGASA變性過程Gibbs自由能變化△G約為3.7 kJ·moL~(-1).相關工作不僅為深入研究融合伴侶Trx對GcGASA變性熱力學、動力學及復性過程影響奠定了基礎;同時,也為通過光譜手段獲取GcGASA的結構信息提供了基礎的數據.Abstract：In the present paper,thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea,desigated as Trx-GcGASA and expressed prokaryotically,was purified and identified by using Ni~(2+)-NTA affinity chromatography column and SDS-PAGE,and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol(DTT),oxidized glutathione(GSSC-),peroxide and guanidine hydrochloride(GdnHC1)by means of steady-state fluorescence spectroscopic methods.It was found that(1)at the neutral Ph Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm,which was ascribed to the fluorescence emission from tyrosine residues.(2)The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8.(3)Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol·L~(-1) GSSG or 5 mmol·L~(-1) peroxide.The latter was roughly consistent with the antioxidative activity reported in vivo.(4)No matter whether 1 mmol· L~(-1) DTT was absent or present,the fusion protein could not be fully unfolded with λ_(max)＜350 nm following the treatment of 6 mol·L~(-1) GdnHCI.(5)Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process,and the unfolding equilibrium curve could be well fitted by using two-state model,giving the Gibbs free energy change(△G)of 3.7 KJ·mol ~(-1).However,it was not the case for reduced Trx-GcGASA protein.The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics,kinetics and refolding process of Trx-GcGASA,but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods. 作者： 張騰[1]馮娟[1]李陽[1]陳銳[1]湯麗霞[1]龐小峰[1]任正隆[2] Author： ZHANG Teng[1]  FENG Juan[1]  LI Yang[1]  CHEN Rui[1]  TANG Li-xia[1]  PANG Xiao-feng[1]  REN Zheng-long[2] 作者單位： 電子科技大學生命科學與技術學院,四川,成都,610054電子科技大學生命科學與技術學院,四川,成都,610054;四川農業大學植物遺傳育種省級重點實驗室,四川,雅安,625014 期 刊： 光譜學與光譜分析   ISTICEISCIPKU Journal： SPECTROSCOPY AND SPECTRAL ANALYSIS 年，卷(期)： 2010, 30(2) 分類號： Q657.3 關鍵詞： 赤雷酸誘導的富含半胱氨酸蛋白    內源熒光    二硫鍵    變性    Keywords： Gibberellin-induced cysteine-rich protein    Intrinsic fluorescence    Disulphide bonds    Denaturation    機標分類號： R73 TQ4 機標關鍵詞： 二硫鍵    氧化還原狀態    Trx    酸誘導    富含半胱氨酸蛋白    內源熒光    熒光及    變性過程    過程影響    Fluorescence    fluorescence intensity    fusion protein    融合蛋白    guanidine hydrochloride    affinity chromatography    SDS-聚丙烯酰胺凝膠電泳    antioxidative activity    鹽酸胍    酪氨酸    unfolding 基金項目： 國家自然科學基金，973計劃項目

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