光谱法表征蛋白酶K的热变性过程

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335 nm红移至354 nm;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 1 ns降低至4.032 4 ns;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。

Characterization of Thermal Denaturation Process of Proteinase K by Spectrometry

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 proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous 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 α-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 α-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.