光诱导辣根过氧化物酶催化活性变化及机理研究

Mechanism of the Variation of Horseradish Peroxidase Catalytic Activity Induced by Light

辣根过氧化物酶(HRP)是广泛应用的一种生物催化剂,其光照后HRP酶活性变化的机理还未见报道。利用紫外-可见(UV-Vis)、同步荧光、圆二色(CD)光谱研究了光对HRP催化活性及酶活变化机制。实验结果表明:光照射HRP可促使其发生还原反应,同时其催化活性会发生变化。UV-Vis吸收光谱数据显示,光照射后HRP的Soret带403 nm最大吸收峰红移、Q带498 nm处氧化峰强度下降,说明经光照射HRPFe(Ⅲ)被还原为HRPFe(Ⅱ)。不同波长对光照还原影响程度为280 nm> 254 nm> 498 nm> 403 nm;酶活为403 nm> 498 nm> 254 nm> 280 nm; 280 nm波长光照射下,Phe,Trp,Tyr和Cys四种游离氨基酸以及谷胱甘肽的加入均对光照还原有促进作用。酶活性与光照还原程度紧密相关,因Fe(Ⅱ)无法提供空轨道与H2O2配位,所以光诱导Fe(Ⅲ)还原导致酶活下降。同步荧光和圆二色光谱揭示了光照还原后HRP血红素的构象发生了变化,构象的变化也是光诱导酶活变化的原因之一。紫外光影响酶活性下降的因素为光照引发活性中心Fe(III)还原,蛋白质构象变化的贡献。研究结果对进一步了解光对含血红素蛋白(酶)结构和功能的影响有重要的理论和实际意义。

Horseradish Peroxidase(HRP)is a widely used biocatalyst.However,activity change mechanism of HRP after irradiation has not been reported.In this research,the effects of irradiation on the catalytic activity of HRP and the mechanism of enzyme activity were studied by UV-Vis absorpti on、synchronous fluorescence and circular dichroism(CD)spectroscopy.The experimental results showed that irradiation could promote HRP reduction,while its catalytic activity would change.The UV-Vis absorption spectra date showed that the maximum absorption peak of the Soret band of HRP was red shift after irradiat ion and the oxidation peak intensity at 498 nm was decreased,and consequently HRPFe(Ⅲ)was reduced to HRPFe(Ⅱ).The effect sequence of wavelength on the photoreduction was 280 nm>254 nm>498 nm>403 nm;the activity of the enzyme was 403 nm>498 nm>254 nm>280 nm;while irradiated by 280 nm light,Phe,Trp,Tyr,Cysfree amino acids and glutathioneall could promote the photoreduction process.Enzyme activity was closely related to the degree of photoreduction,and Fe(Ⅱ)could not provide empty orbit coordination with H2O2,so light-induced reduction of Fe(Ⅲ)led to decreased activity.Synchrotron fluorescence and circular dichroism spectroscopy revealed that the conformation of HRP did changed after photoreduction,so the conformational change was the reason for the change of enzyme activity.The factors that affect the decrease of enzyme activity by ultraviolet light are the contribution of light-induced active center Fe(Ⅲ)reduction and protein conformational changes.The results of this study have important theoretical and practical significance for a further understanding of the effects of light on hemoglobin(enzyme)structures and functions.