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二硫键的氧化还原状态对Trx融合赤霉酸诱导富含半胱氨酸蛋白内源荧光及变性过程影响
摘要：在采用亲和层析、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对原核表达的赤霉酸诱导的富含半胱氨酸蛋白(Trx-GcGASA)进行纯化、鉴定的基础上,运用稳态荧光光谱手段研究了二硫苏糖醇(DTT)、氧化型谷胱甘肽(GSSG)、过氧化氢、盐酸胍(GdnHCl)对Trx-GcGASA内源荧光及变性过程的影响,发现(1)在中性缓冲体系中融合蛋白的内源荧光以305 nm的酪氨酸的.荧光发射为主;(2)伴随着二硫键还原,融合蛋白中色氨酸和酪氨酸的相对荧光强度比值从0.7变化至1.8倍左右;(3)经过0.5 mmol·L~(-1) GSSG、5 mmol·L~(-1)过氧化氢处理后,酪氨酸和色氨酸的荧光强度下降约12～21%;(4)无论是否采用1 mmol·L~(-1) DTT处理,6 mol·L~(-1)盐酸胍均不能诱导融合蛋白彻底变性;(5)二硫键的存在与否影响了盐酸胍诱导的变性过程.通过两态模型拟合获得Trx-GcGASA变性过程Gibbs自由能变化△G约为3.7 kJ·moL~(-1).相关工作不仅为深入研究融合伴侣Trx对GcGASA变性热力学、动力学及复性过程影响奠定了基础;同时,也为通过光谱手段获取GcGASA的结构信息提供了基础的数据.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： 张腾[1]冯娟[1]李阳[1]陈锐[1]汤丽霞[1]庞小峰[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] 作者单位： 电子科技大学生命科学与技术学院,四川,成都,610054电子科技大学生命科学与技术学院,四川,成都,610054;四川农业大学植物遗传育种省级重点实验室,四川,雅安,625014 期 刊： 光谱学与光谱分析 ISTICEISCIPKU Journal： SPECTROSCOPY AND SPECTRAL ANALYSIS 年，卷(期)： 2010, 30(2) 分类号： Q657.3 关键词： 赤雷酸诱导的富含半胱氨酸蛋白 内源荧光 二硫键 变性 Keywords： Gibberellin-induced cysteine-rich protein Intrinsic fluorescence Disulphide bonds Denaturation 机标分类号： R73 TQ4 机标关键词： 二硫键 氧化还原状态 Trx 酸诱导 富含半胱氨酸蛋白 内源荧光 荧光及 变性过程 过程影响 Fluorescence fluorescence intensity fusion protein 融合蛋白 guanidine hydrochloride affinity chromatography SDS-聚丙烯酰胺凝胶电泳 antioxidative activity 盐酸胍 酪氨酸 unfolding 基金项目： 国家自然科学基金，973计划项目【二硫键的氧化还原状态对Trx融合赤霉酸诱导富含半胱氨酸蛋白内源荧光及变性】相关文章：
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