Zhang Qi-Bing, Na Xin-Zhu, Yin Zong-Ning
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
Guang Pu Xue Yu Guang Pu Fen Xi. 2013 Jul;33(7):1749-53.
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 degrees C, 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 alpha-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 alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.
研究了不同温度对蛋白酶K活性及构象变化的影响。方法：对蛋白酶K进行不同温度处理，然后用变性的天然底物酪蛋白测定酶活性，用稳态和时间分辨荧光光谱研究三级结构，用圆二色性研究二级结构。结果表明，随着温度从25℃升至65℃，蛋白酶K的酶活性和半衰期下降，最大发射波长从335nm红移至354nm，荧光强度降低。色氨酸残基的同步荧光强度降低，酪氨酸残基的同步荧光强度增加。色氨酸残基的荧光寿命从4.427 1ns降至4.032 4ns，α-螺旋比例下降。得出结论：用稳态/时间分辨荧光光谱和圆二色性研究蛋白酶K的热稳定性简单准确。蛋白酶K的热变性遵循三态过程。蛋白酶K的荧光强度受酪氨酸向色氨酸残基的荧光共振能量转移影响。α-螺旋是维持蛋白酶K酶活性部位构象稳定性的主要结构。
