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  

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

作　　者：何海伦[1] 陈秀兰[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,山东济南250100

出　　处：《山东大学学报（理学版）》2013年第1期23-29,42,共8页Journal of Shandong University(Natural Science)

基　　金：国家863计划海洋生物技术主题课题资助项目(2010AA09Z404);国家自然科学基金资助项目(31070061);海洋公益资助项目(201005032-6)

摘　　要：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-01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1.0 mol/L和3.0 mol/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<Tris-HCl缓冲液<硼酸缓冲液;而中温蛋白酶BP-01在上述3种缓冲液中保温并没有表现出构象的明显变化。上述结果表明,适冷蛋白酶MCP-01结构具有较强的柔性和较低的稳定性,比中温蛋白酶BP-01更容易受到变性剂和溶液环境的影响。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 BP4）I, which led to its higher fexibility 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 （GuHC1） 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 GuHC1 for MCP-01 and BP431 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 concen- tration was further increased, the structure of MCP-01 and BP-01 began to denature. These results indicated that the sta- bility of MCP-01 was less than that of BP-O1 and it was denatured at lower GuHCI or TFE concentration. The confor- mation changes of protease MCP-01 and BP-01 were studied during incubation. The stability of MCP431 was affected significantly by different buffer system and increased successively in the following order： carbonate 〈 Tris-HCI 〈 bo- rate. But the stability of BP-01 was same in different buffer. These results suggested that the structure of cold-adapted protease MCP-01 was higher flexibility and lower stability, so it was more vulnerable to denaturant or solution environ- ment than mesophilic protease BP-01.

关 键 词：适冷蛋白酶 中温蛋白酶 圆二色光谱 构象 

分 类 号：Q93[生物学—微生物学]