Effects of pH Value and Mg2+ Concentration on Bioactivity and Structure of Trypsin

• Main Authors: Pen-Yuan Chen, 陳本源
• Other Authors: Fu-Yung Huang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/emum5s

碩士 === 國立成功大學 === 化學系碩博士班 === 92 === 　　In this study, we observed that the existence of Mg2+ had substantial effects on the catalytic hydrolysis of trypsin toward N-benzoyl-L-arginine ethyl ester (BAEE), however, the variations of pH ( pH 7, 8, and 9) value showed little effect on the activity. In the presence of 0.05 M Mg2+, the hydrolytic initial rate increased 40%, while the concentration of Mg2+ was 1.5 M, the initial rate decreased 14%. In order to investigate the structural changes of trypsin in the presence of Mg2+ and the variations of pH values, CD and Fluorescence spectra were measured. Far-UV CD spectra showed that with the presence of Mg2+ the percentage of 2o structural component of α-helix increased with the increasing of Mg2+ concentration. And the variation of pH showed no effect on the secondary components. Near-UV CD spectra indicated the tertiary structure of trypsin had little changes, suggesting the binding of Mg2+ might result in minor local alteration, which made little change on the overall structure. Trptophan emission spectra showed there had 50% increase in intensity with the presence of 2 M Mg2+ and the maximum emission wavelength did not shift, indicating no obvious environmental change around the tryptophan resideues. ANS fluorescence spectra showed when the concentration of Mg2+ was 1.5 M and 2.0 M, the intensity increased up to 1.5 and 1.7 fold, respectively, compared with that without the presence of Mg2+, indicating the hydrophobic region increased upon the addition of Mg2+. MIANS fluorescence measurements showed that there were 56% loss in the intensity with the presence of 2 M Mg2+, suggesting that the overall structure change had caused cysteine residues embedded and simultaneously more tryptophan and aromatic side chain containing amino acid residues exposed. This study has displayed that the hydrolytic activity of trypsin is dependent on the concentration of Mg2+.