变性剂和缓冲系统对适冷蛋白酶MCP-01和中温蛋白酶BP-01构象影响的圆二色光谱分析何海伦， 陈秀兰*

运用圆二色（CD）光谱分析了适冷蛋白酶MCP-01和中温蛋白酶BP-的二级结构。与BP-01相比，MCP-01具有较低的α螺旋含量和较高的无规则卷曲，表明适冷蛋白酶MCP 01的结构可能具有较高的柔性和较低的稳定性。测定MCP-01和BP-01的变性温度表明，MCP-01的变性温度比BP 01 低10.7℃。测定了变性剂盐酸胍（GuHCl）、2，2，2 三氟乙醇（TFE）对MCP-01和BP-01结构的影响。盐酸胍使MCP-01和BP 01变构的浓度分别为1.0mol/L和3.0mol/L，TFE造成MCP 01和BP 01开始变构的含量分别为20%和30%。这表明，与中温酶相比，变性剂在较低浓度下就可使适冷酶变构，从而造成活性的丧失。检测了两种蛋白酶在保温过程中构象的变化情况。不同缓冲系统明显影响MCP 01的构象稳定性，MCP 01在不同缓冲液中构象稳定性为：碳酸缓冲液

Circular dichroism detection of the effects of denaturants and buffers on the conformation of cold-adapted protease MCP-01 and mesophilic protease BP-01

The secondary structure of cold-adapted protease MCP-01 and mesophilic protease BP-01 were investigated by circular dichroism (CD). According to the result of CD spectra, there are lower content of α-helixes and higher content of random in MCP 01 than in BP-01, which led to its higher flexibility and lower stability in structure. The temperature leading to loss of 50% secondary structure of protease MCP-01 was lower 10.7℃ than BP-01. The effects of denaturant guanidine hydrochloride (GuHCl) and 2,2,2 Trifluoroethanol (TFE) on cold-adapted protease MCP-01 and mesophilic protease BP-01 were studied. The results of CD spectra indicated that the unfolding concentration of GuHCl for MCP-01 and BP-01 were 1.0 M and 3.0 M， respectively. Moreover, the CD spectra showed that the structure of MCP-01 and BP-01 had no obvious changes when TFE concentrations were lower 20% and 30%, respectively. When TFE concentration was further increased， the structure of MCP-01 and BP-01 began to denature. These results indicated that the stability of MCP-01 was less than that of BP-01 and it was denatured at lower GuHCl or TFE concentration. The conformation changes of protease MCP-01 and BP-01 were studied during incubation. The stability of MCP-01 was affected  significantly by different buffer system and increased successively in the following order: carbonate