Effects of pH Value and Zn2+ Concentration on bioactivity and structure of Trypsin

• Main Authors: Chun-Chi Li, 李俊奇
• Other Authors: Fu-Yung Huang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/e75zg6

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 Zn2+ concentration on the bioactivity and structure of trypsin. It was found that the existence of the Zn2+ is capable of affecting the hydrolytic ability of trypsin on the substrate of N-benzoyl arginine ethyl ester (BAEE). The inhibition of Zn2+ on trypsin is uncompetitive binding at a site different from that of BAEE. When [Zn2+] = 166 mM and at pH 7.16, the initial hydrolytic rate toward BAEE was dropped 33.57%; furthermore, with the same concentration of Zn2+and pH 6.5, it was found that 42.92% of the activity was lost. In order to investigate the structural change of trypsin under this condition, CD and Fluorescence spectra were measured. Far-UV CD spectra showed that the existence of Zn2+ caused the percentage of 2o structural component of α-helix increased with the increasing of Zn2+ 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 did not show obvious changes, except little decreasing of intensity, suggesting the structural changes caused due to the binding of Zn2+ was a local alteration. ANS, Trptophan and MIANS fluorescence spectra showed that the hydrophobic region increased with the existence of Zn2+, indicative of more tryptophan resides and aromatic side chain containing amino acid residues exposed; however, when pH value was in acidic condition, the hydrophobic region was decreased, suggesting low pH value is not a good condition for trypsin hydrolytic activity.