水合和pH对蛋白酶K热诱发转变影响的量热法研究

用差示扫描量热法, 在310～450 K温度范围、0.048～4.39克水/克干酶以及pH3～9范围内研究了蛋白酶K(EC 3.4.21.14)的热变性。分别提出表征热变性过程的三个主要热力学参数: 热变性温度T_d、比变性焓△H_d及超额表观比热C_(ex)~(max)和总水量h以及pH的关系。除Td和h的关系属于Flory-Garrett类型外, 蛋白酶K的热变性热力学特征和已报道的球蛋白、双螺旋蛋白及三螺旋蛋白不相同。一级水合水对蛋白酶K热变性热力学特征有最大的影响, pH也是影响该酶热稳定性的重要因素。

CALORIMETRIC STUDY OF THE INFLUENCE OF HYDRATION AND pH UPON THE THERMALLY-INDUCED CONFORMATIONAL TRANSITION OF PROTEINASE K

Differential scanning calorimetry was used to investigate the thermal denaturation of the proteinase K (EC 3.4.21.14) in the temperature range from 310 to 450 K. The influence of hydration and pH upon the thermal denaturation was studied in the range of total water content from 0.048 to 4.39 g H_2O/g dry enzyme and in the pH range from 3.29 to 8.49, respectively. The dependence of the denaturational temperature T_d, the specific enthalpy of denaturation ΔH_d and the maximum of excess apparent specific heat C_ex~(max) upon the degree of hydration h and the pH of the buffer is presented respectively. The relationship between the denaturational temperature T_d and the water content is of the Flory-Garrett's type. Besides this, the thermodynamic characteristics of the thermal denaturation of proteinase K are different from those reported of globular proteins, double-stranded coiled-coils and triple-stranded coiled-coils. The influence of primary hydration water upon the thermodynamic parameters of the thermal denaturation is the greatest. The external variable, the pH of the buffer used to dissolve the enzyme influences its thermostability to some extent.