Goyal Monu Kumari, Roy Ipsita, Banerjee Uttam Chand, Sharma Vikas Kumar, Bansal Arvind Kumar
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Punjab, India.
Eur J Pharm Biopharm. 2009 Feb;71(2):367-76. doi: 10.1016/j.ejpb.2008.09.012. Epub 2008 Oct 2.
The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 degrees C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.
本研究的目的是调查模型蛋白溶菌酶在常用防腐剂苯甲醇存在下的稳定性。采用溶菌测定、尺寸排阻色谱、圆二色性、差示扫描量热法、天然聚丙烯酰胺凝胶电泳和动态光散射等技术，研究了苯甲醇存在下溶菌酶的整体稳定性。在苯甲醇存在下，溶菌酶对热应激的稳定性更高。与对照样品22%的活性相比，在含有0.5%、0.9%和2%（v/v）苯甲醇的情况下，酶在75℃下暴露2小时后，分别显示出33%、42%和75%的残余活性。由于单体浓度的损失，相对于含有苯甲醇的样品，对照样品中观察到聚集体尺寸逐渐增加。发现这种效应具有浓度依赖性，2%的苯甲醇对热诱导的解折叠和聚集具有最大的预防作用。这种效应很显著，因为在苯甲醇存在下酶的热转变温度会降低。苯甲醇有利于溶菌酶的热变性，但能稳定溶菌酶以防止热诱导的聚集。
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