鎂離子含量的多寡以及pH值的不同對胰蛋白酶的催化活性和結構有程度上的影響；在催化活性上當鎂離子濃度在0.05 M時，其初始速率和無Mg2+相比明顯增加約有40%；而當鎂離子濃度在1.5 M的情況下，相較於無Mg2+水解初始速率則變慢約14%。在結構上的影響，鎂離子的存在會使得Far-UV CD光譜圖有紅位移的現象，顯示出二級結構成份的含量有變化。結構的細微變化顯示出胰蛋白酶Trp螢光，在加入鎂離子後已有所改變，原因是鎂離子的加入(2.0 M)改變了胰蛋白酶的構形使得內部疏水性的Trp被暴露，因此所偵測到的Trp螢光變強約50%，但最大放射波長並沒有位移產生，這表示在Trp殘基的環境上沒有明顯地改變。當鎂離子濃度增加時(0.05 M)，其ANS特性螢光有增強的趨勢，這顯示出隨著鎂離子濃度的增加，胰蛋白酶表面的疏水性區域也隨之增加；若鎂離子的濃度高於1 M時(1.5 M、2.0 M)，不但ANS螢光強度分別增強為無Mg2+的1.5、1.7倍，而且其最大放射波長有很明顯往短波長位移，象徵著ANS所結合的區域轉變成更具疏水性。MIANS光譜的測量顯示出在鎂離子濃度高時(2.0 M)比無Mg2+，MIANS螢光強度下降56%，代表在高含量(2.0M)鎂離子狀態下，MIANS和半胱胺酸(Cysteine)殘基的SH-group鍵結數量變少，而導致螢光強度下降。

　　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+.

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