化学学报 ›› 2010, Vol. 68 ›› Issue (02): 194-198. 上一篇    下一篇

研究简报

等转化率法分析溶菌酶在添加剂影响下的热变性行为

曹小敏*,1,王志勇2,刘欲文2,汪存信2,田耘3   

  1. (1长沙环境保护职业技术学院 湖南 长沙 410004)
    (2武汉大学化学与分子科学学院 湖北 武汉 430072)
    (3湖南省环境监测中心站 湖南 长沙 410014)
  • 投稿日期:2009-06-05 修回日期:2009-08-02 发布日期:2010-03-17
  • 通讯作者: 曹小敏 E-mail:caograss2003@yahoo.com.cn
  • 基金资助:

    国家自然科学基金(No. 20373050 and 30600116)

Effect of Additive on the Thermal Denaturation of Lysozyme Analyzed by Isoconversional Method

Cao Xiaomin*,1 Wang Zhiyong2 Liu Yuwen2 Wang Cunxin2 Tian Yun3   

  1. (1Changsha Environmental Protection College, Changsha 410004)
    (2College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072)
    (3Environmental Monitoring Center of Hunan Province, Changsha 410014)
  • Received:2009-06-05 Revised:2009-08-02 Published:2010-03-17
  • Contact: CAO Xiao-Min E-mail:caograss2003@yahoo.com.cn

利用差示扫描量热法(DSC)研究了溶菌酶在不同浓度的添加剂(蔗糖、葡萄糖、果糖、甘露醇)和不同浓度的磷酸盐缓冲液影响下的热变性过程, 并用等转化率法对该过程进行了分析. 变性温度Tm随扫描速率和添加剂浓度的增加而提高, 随磷酸盐缓冲液浓度的增加而降低. 磷酸盐加速了溶菌酶的变性过程, 降低了溶菌酶的热稳定性, 而添加剂则增强了溶菌酶的热稳定性. 在添加剂存在的条件下, 溶菌酶变性过程部分为聚集不可逆过程, 部分则可逆. 等转化率法表明溶菌酶在所有条件下, 其表观活化能在不同的转化率下并未保持不变, 而是随转化率增大而减小, 说明了一个简单的反应机理并不能用来描述溶菌酶的变性过程, 其过程并不是标准两态可逆过程, 而是一个涉及多种蛋白质状态的复杂过程.

关键词: 差示扫描量热法, 溶菌酶, 添加剂, 等转化率法, 复杂过程

The thermal denaturation of lysozyme was studied as a function of different additive (sucrose, glucose, fructose, mannitol) concentrations, phosphate buffer concentration and scan rate using differential scanning calorimetry (DSC ) and analyzed by an isoconversional method. The denaturation temperature Tm increased with the increase of scan rate and the additive concentrations, while decreased with the increase of phosphate concentration. The phosphate accelerated the denaturation process of lysozyme and reduced the thermal stability of lysozyme, while the additives enhanced it. One part of the lysozyme denaturation process was irreversible in which aggregation occurred, and the other part was reversible in the presence of all additives. By the isoconversional method, it was showed that the apparent activation energy did not keep invariable at different conversion ratios (α) under all conditions, but decreased with the increase of α, which suggested that a simple reaction mechanism could not be used for the denaturation process. The denaturation was still not a process involving the two standard reversible states, but a multi-step process in the presence and the absence of all additives.

Key words: DSC, lysozyme, additive, isoconversional method, multi-step process