Mehta Ranjana, Kundu Agnita, Kishore Nand
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
Int J Biol Macromol. 2004 Apr;34(1-2):13-20. doi: 10.1016/j.ijbiomac.2003.11.001.
The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.
在4-氯丁醇存在的情况下,通过差示扫描量热法研究了核糖核酸酶A的热变性。在低浓度(高达50 mM)的该醇存在下,于pH 5.5观察到热转变是可逆的;在中等浓度(50 mM < c < mM)时是不可逆的;而在250 mM及更高浓度的4-氯丁醇存在下又变为可逆。在50 mM 4-氯丁醇存在时,核糖核酸酶A以两种构象状态存在,在不同温度下展开。可逆热转变已被拟合为两态天然态到变性态的机制。不可逆热转变已根据两态不可逆天然态到变性态动力学模型进行分析。使用不可逆模型,计算了速率常数作为温度的函数以及不可逆过程的活化能。圆二色性和荧光光谱结果证实了差示扫描量热法的观察结果,并表明在对应于第二个转变的温度下,在50 mM