神经红蛋白突变体H64V与叠氮离子的作用
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摘要
将64位组氨酸(His64)突变为缬氨酸(Val),构建了神经红蛋白(Ngb)突变体H64V基因并进行了蛋白的表达纯化,突变使Ngb的血红素Fe由六配位结构变成五配位结构.利用光谱法研究了突变体H64V与叠氮离子(N_3~-)的相互作用.紫外-可见吸收光谱表明,H64V与N_3~-键合后其Soret带吸收峰由406 nm红移至418 nm;荧光光谱表明N_3~-可使H64V的荧光发生静态猝灭.计算得到了H64V与N_3~-键合的平衡解离常数和热力学参数.另外,还研究了不同阴离子对H64V与N_3~-结合的影响,发现阴离子的存在减弱了N_3~-与H64V之间的结合力.
Mutating the histidine 64 to valine,the gene of Neuroglobin(Ngb) mutant H64V was constructed and its protein was expressed and purified.This mutation changed the coordination geometry of neuroglobin from hexa-to penta-coordinated.The interaction of neuroglobin mutant H64V with azide anion(N_3~-) was investigated by spectroscopy.UV-Vis absorbance spectra showed that the binding of mutant H64V to N_3~- induced the change of the Soret-band absorption peak from 406 nm to 418 nm.Fluorescence spectra indicated that the fluorescence of mutant H64V was quenched by N_3~- through static quenching mechanism.The binding equilibrium dissociation constant and thermodynamic parameters between mutant H64V and N_3~- were calculated.In addition,the effects of different anions on their binding were studied,the results showed that the anions reduced the binding ability between mutant H64V and azide anion.
Mutating the histidine 64 to valine,the gene of Neuroglobin(Ngb) mutant H64V was constructed and its protein was expressed and purified.This mutation changed the coordination geometry of neuroglobin from hexa-to penta-coordinated.The interaction of neuroglobin mutant H64V with azide anion(N_3~-) was investigated by spectroscopy.UV-Vis absorbance spectra showed that the binding of mutant H64V to N_3~- induced the change of the Soret-band absorption peak from 406 nm to 418 nm.Fluorescence spectra indicated that the fluorescence of mutant H64V was quenched by N_3~- through static quenching mechanism.The binding equilibrium dissociation constant and thermodynamic parameters between mutant H64V and N_3~- were calculated.In addition,the effects of different anions on their binding were studied,the results showed that the anions reduced the binding ability between mutant H64V and azide anion.
引文
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[12]Maurus R.,Bogumil R.,Nguyen N.T.,Mauk A.G.,Brayer G.,Biochem.J.,1998,332(1),67—74
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[19]Li L.Z.,Ji H.W.,Zhao C.,Xu T.,Liu G.F.,Fu C.L.,Yan H.C.,Chinese Sci.Bull.,2005,50(10),964—969(李连之,冀海伟,赵超,许涛,刘国富,付崇罗,闫华超.科学通报,2005,50(10),964—969)
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