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山东大学学报(理学版) ›› 2013, Vol. 48 ›› Issue (1): 23-29.doi: 10.6040/j.issn.1671-9352.0.2012.429

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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*

何海伦, 陈秀兰*   

  1. 山东大学微生物技术国家重点实验室, 山东  济南  250100
  • 收稿日期:2012-08-21 出版日期:2013-01-20 发布日期:2013-01-15
  • 通讯作者: 陈秀兰(1966- ),女,教授,博士,主要从事海洋微生物技术研究.Email: cxl0423@sdu.edu.cn
  • 作者简介:何海伦( 1976- ),女, 副教授, 博士,主要从事海洋微生物酶学研究. Email: helenhe@sdu.edu.cn
  • 基金资助:

    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)

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

HE Hai-lun,  CHEN Xiu-lan*   

  1. The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100,  Shandong,  China
  • Received:2012-08-21 Online:2013-01-20 Published:2013-01-15

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-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

关键词: 中温蛋白酶, 构象, 适冷蛋白酶, 圆二色光谱

Abstract:

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

Key words: conformation, circular dichroism, cold-adapted protease, mesophilic protease

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