JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE) ›› 2013, Vol. 48 ›› Issue (1): 23-29.doi: 10.6040/j.issn.1671-9352.0.2012.429

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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


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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