Gheibi N, Saboury A A, Haghbeen K, Moosavi-Movahedi A A
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
J Biosci. 2006 Sep;31(3):355-62. doi: 10.1007/BF02704108.
The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.
研究了双孢蘑菇酪氨酸酶（MT）在10 mM磷酸盐缓冲液（pH 6.8）中，于4℃和40℃两个温度下储存时，在三种不同氨基酸（组氨酸、苯丙氨酸和天冬氨酸）以及作为渗透剂的海藻糖存在下的热力学稳定性和剩余活性，以便与未添加任何添加剂时获得的结果进行比较。失活动力学符合一级定律。失活速率常数（kinact）值是描述渗透剂对酶动力学稳定性影响的最佳参数。与不存在时（2.5×10⁻⁴ s⁻¹）相比，海藻糖和组氨酸的kinact值最小（0.7×10⁻⁴ s⁻¹）。此外，为了获得这四种渗透剂对酶热力学稳定性的影响，研究了十二烷基三甲基溴化铵（DTAB）引起的蛋白质变性和热扫描。根据佩斯理论的二态模型分析S形变性曲线，以确定室温下水溶液中变性过程的吉布斯自由能变化，这是表明蛋白质稳定性的一个非常好的热力学标准。尽管组氨酸、苯丙氨酸和天冬氨酸导致MT三级结构收缩，但其二级结构没有任何变化，结果是MT的化学和热稳定性提高。在海藻糖存在下，该酶的热力学和结构变化表现出良好的一致性。因此，在这四种渗透剂中，海藻糖是一种特殊的蛋白质稳定剂。
