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  

Effects of Redox State of Disulfide Bonds on the Intrinsic Fluorescence and Denaturation of Trx-Fused Gibberellin-Induced Cysteine-Rich Protein from Gymnadnia conopsea

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[1,2] 

机构地区：[1]电子科技大学生命科学与技术学院,四川成都610054 [2]四川农业大学植物遗传育种省级重点实验室,四川雅安625014

出　　处：《光谱学与光谱分析》2010年第2期395-400,共6页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(30730065;20973034);(973计划)项目(2007CB936103)资助

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-1GSSG、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-1DTT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ΔG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。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 Ni2＋-NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol （DTT）, oxi- dized glutathione （GSSG）, 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 excita- tion at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine resi- dues. （2） The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryp- tophan 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 re- ported in vivo. （4） No matter whether 1 mmol·L-1 DTT was absent or present, the fusion protein could not be fully unfolded with λmax〈50 nm following the treatment of 6 mol · L -1 GdnHC1. （5） Fusion protein Trx-GcGASA experienced GdnHCl-in- duced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change （ΔG） of 3. 7 kJ · mol- 1. However, it was not the case for reduced Trx-GeGASA protein. The aforemen- tioned 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-GeGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

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 

分 类 号：Q657.3[生物学—生物物理学]