Effects of pH Values and Mg2+ Concentration on bioactivity and structure of α-Chymotrypsin

• Main Authors: Shiao-Yen Shih, 施曉燕
• Other Authors: Fu-Yung Huang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/93484915271397796458

碩士 === 國立成功大學 === 化學系碩博士班 === 92 === 　　Circular Dichroism (CD) and fluorescence Spectroscopy have been used to investigate the effects of pH value and Mg2+ concentration on the bioactivity and structure of α-chymotrypsin. It was found that the existence of the Mg2+ was capable of affecting the hydrolytic ability of α-chymotrypsin on the substrate of N-benzoyl-L-tyrosine ethyl ester (BTEE). When [Mg2+] = 103 mM and at pH 8.0, the initial hydrolytic rate toward BTEE was dropped 37% compared with that of free Mg2+; furthermore, when [Mg2+] was 620 mM and at the same pH, it was found that 52% of the activity was lost. Further investigation showed that the more deviation of pH from 8, the more loss of the activity. At pH 7.5 and pH 7.0 the activity decreased 21% and 27%, respectively, compared with that obtained at pH 8. In order to investigate the structural changes of α-chymotrypsin under this condition, CD and Fluorescence spectra were measured. Far-UV CD spectra showed that with the existence of Mg2+ the percentage of 2o structural component of α-helix increased with the increasing of Mg2+ concentration. And the deviation of pH from 8 also resulted in the same results. However, the Near-UV CD spectra indicated the tertiary structure of trypsin had little changes, suggesting the structural change caused due to the binding of Mg2+ might be a local alteration. ANS, Trptophan and MIANS fluorescence spectra showed that the hydrophobic regions decreased with the existence of Mg2+, indicative of the alteration of structure resulted in some of the already exposed tryptophan residues and aromatic side chain containing amino acid residues were buried inside; however, the change of pH value had caused little alterations of tertiary and secondary structures, suggesting pH had little effects on the α-chymotrypsin hydrolytic activity compared with that caused by the existence of Mg2+.