诱导蛋白酶K去折叠过程的热力学研究

Study on thermodynamics of the induced unfolding process of proteinase K

目的研究诱导蛋白酶K的去折叠过程,尝试阐明构象与酶活力的关系。方法蛋白酶K经过盐酸胍处理后,以变性酪蛋白为底物测定酶活力,采用荧光光谱法研究酶分子的构象变化,计算相关的热力学参数。结果随着盐酸胍浓度升高,蛋白酶K的酶活力和荧光强度降低,峰位红移,碘化钾对酶分子的内源性荧光猝灭作用增强,去折叠分数增大;蛋白酶K的热变性中点温度和最大稳定温度降低;去折叠过程的熵变大于0。结论蛋白酶K对低浓度的盐酸胍较为稳定,3.5 mol·L-1为盐酸胍诱导蛋白酶K变性的临界浓度;空间构象的稳定性是维持酶活力的基础;蛋白酶K的热稳定性随盐酸胍浓度增大而降低;盐酸胍诱导蛋白酶K的去折叠过程是熵驱动的。

OBJECTIVE To study the induced unfolding process of proteinase K and the relationship between conformation and activity. METHODS Proteinase K was treated with different concentrations of guanidine hydrochloride （ Gu）, and then denatured sub- strate casein was used to assay enzyme activity. Fluorescence spectroscopy was used to study the eonformational changes, and the ther- modynamic parameters of unfolding process were calculated. RESULTS As the concentration of Gu rised, the enzyme activity and flu- orescence intensity of proteinase K decreased, maximum emission wavelength red shifted, and an attenuation of the Stern - Volmer quenching constant of potassium iodide and the unfolding fraction were observed. The midpoint of thermal denaturation and the tempera- ture of maximum stability reduced. CONCLUSION Proteinase K was stable to the low concentrations of Gu. The critical concentration of Gu in the unfolding process of proteinase K was 3.5 tool ＂L-1. Conformational stability was the maintenance of enzyme activity. The thermal stability of proteinase K decreased with the concentration of Gu increased. The unfolding process of proteinase K induced by Gu was driven by entropy.