光诱导高铁肌红蛋白还原的光谱法研究

Spectral Study on the Photoreduction of Metmyoglobin

采用紫外-可见吸收、荧光及圆二色(CD)等光谱法为主要分析手段,对光诱导高铁肌红蛋白(metmyoglobin,metMb)还原及其引起的蛋白结构变化进行了研究.采用氙灯430 nm的单色光和紫外灯对高铁肌红蛋白溶液进行光照实验,实验结果显示,在metMb的Q吸收带545 nm和580 nm附近吸收峰强度显著增强.通过验证性实验,证明光照后metMb发生了还原反应.验证性实验包括:metMb中加入还原剂连二亚硫酸钠实验;光照后样品加入氧化剂铁氰化钾的可逆性实验;光照的同时通入CO实验.进一步讨论表明,采用氙灯430 nm的单色光诱导metMb还原的机理为光诱导的分子内电子转移.此外,对光还原反应后的样品进行同步荧光和CD光谱分析,发现蛋白质色氨酸残基微环境极性增加,α-螺旋的含量由63%下降到57%,而β-折叠的含量增加了7%.

The photoreduction of metmyoglobin（metMb） and its effects on the changes of protein structure were investigated by UV-visible absorbance,fluorescence and circular dichroism（CD） spectra.MetMb in Na2HPO4-NaH2PO4 buffer were irradiated with xenon lamp at 430 nm and ultraviolet lamp.The results in-dicated that the absorbance intensity at Q band（545 nm and 580 nm） increased evidently after illumination.Through experimental verification,metMb had been reduced after the illumination.The verifying experi-ment included： adding sodium dithionite to metMb;adding potassium ferricyanide to the sample after the il-lumination;irradiating meanwhile adding CO to the sample.Through further discussion,it can be seen that the photoreduction mechanism of metMb with xenon lamp at 430 nm was photoinduced intramolecular elec-tron transfer.In addition,the results of synchronous fluorescence and CD spectra of the sample after irradia-tion suggested that the polarity of microenvironment of tryptophan residues increased and the secondary structure of protein（CD data） varied with α-helices reduction from 63% to 57%,and β-sheet increase by 7%.