稳态荧光光谱法分析超氧化物岐化酶溶液的稳定性

Analysis of Stability of Superoxide Dismutase Solution by Steady-state Fluorescence Spectroscopy

目的考察超氧化物岐化酶(SOD)溶液在不同影响因素下的活性及构象变化。方法通过邻苯三酚自氧化速率法测定SOD溶液的活性,利用稳态荧光光谱法分析在不同影响因素(pH值、超声、变性剂盐酸胍)下SOD三、四级结构的变化与荧光光谱特征变化的关系。结果溶液pH值降低,SOD稳定性增加;在pH2.2～pH7.0的范围内,pH值与SOD比活力呈线性相关;随着pH值的降低,普通荧光最大发射波长由336nm蓝移至325nm。超声次数的增加使同步光谱谱带蓝移,SOD活性下降。随着盐酸胍浓度的升高,SOD活性逐渐下降;在盐酸胍浓度达2.1mol/L时,SOD最大荧光发射峰蓝移至312nm;经盐酸胍变性后,酶的荧光强度比天然态酶有所增加。结论采用稳态荧光光谱法可分析SOD溶液的稳定性。

Objective To investigate the effects of various influencing factors on activity and conformation of superoxide dismutase（SOD）. Methods The activity of SOD solution was determined by pyrogallol auto-oxidation rate method. The relationship between the changes of fluorescence spectrum and those of tertiary and quarternary structures of SOD in presence of various influencing factors（pH value, ultrasonication and guanidine hydrochloride as denaturant）was analyzed by steady-state fluorescence spectroscopy. Results The stability of SOD increased with the decreasing pH value. The pH value at a range of 2. 2 ～ 7. 0 was linearly related to specific activity of SOD. With the decreasing pH value, the maximum emission wavelength of general fluorescence showed a blue shift from 336 nm to 325 nm. The increased times of ultrasonication caused the blue shift of synchronous spectrum bands and the decrease of SOD activity. The SOD activity decreased gradually with the increasing guanidine hydrochloride concentration. When the guanidine hydrochloride concentration reached 2. 1 mol / L, the maximum fluorescent emission peak showed a blue shift to 312 nm. The fluorescence intensity of SOD de-naturalized with guanidine hydrochloride increased as compared with that of natural SOD. Conclusion It is feasible to analyze the stability of SOD by steady-state fluorescence spectroscopy.