光谱法表征蛋白酶K的热变性过程  

Characterization of Thermal Denaturation Process of Proteinase K by Spectrometry

作　　者：张奇兵[1] 那馨竹[1] 尹宗宁[1] 

机构地区：[1]四川大学华西药学院靶向药物与释药系统教育部重点实验室,四川成都610041

出　　处：《光谱学与光谱分析》2013年第7期1749-1753,共5页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(30973659)资助

摘　　要：用不同温度处理蛋白酶K,以变性酪蛋白底物法测定酶活力,稳态/瞬态荧光光谱法和圆二色谱法测定空间构象和二级结构,研究温度对蛋白酶K酶活力和构象的影响。温度由25℃升高至65℃过程中,蛋白酶K的酶活力逐渐降低,半衰期缩短;发射光谱荧光强度降低,峰位由335nm红移至354nm;色氨酸残基同步荧光强度降低,酪氨酸残基同步荧光强度增大;色氨酸残基荧光寿命由4.427 1ns降低至4.032 4ns;α-螺旋百分含量降低。结果表明:采用稳态/瞬态荧光光谱法和圆二色谱法能较简便、准确的描述蛋白酶K的热稳定性变化;蛋白酶K的热变性过程符合三态模型,存在一个中间态;蛋白酶K分子内部存在酪氨酸残基对色氨酸残基的共振能量转移作用;α-螺旋是维系蛋白酶K活性中心构象稳定性的主要结构。The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65℃, the enzyme activity and half-life of pro- teinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Syn- chronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of a-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The a-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

关 键 词：蛋白酶K 热稳定性 稳态荧光光谱 瞬态荧光光谱 圆二色谱 

分 类 号：Q55[生物学—生物化学]