在此篇論文中，紫外線光譜、螢光光譜 (包含：色胺酸螢光光譜、非色胺酸螢光光譜、ANS螢光光譜、MIANS螢光光譜) 及圓二色光譜 (包含：遠紫外光-圓二色光譜、近紫外光-圓二色光譜) 被用來探討不同pH值和不同鎂離子濃度對α-胰凝乳蛋白酶在活性上及構形上的影響。實驗結果顯示出，鎂離子存在時會影響α-胰凝乳蛋白酶之活性，在pH值為8.0，鎂離子濃度為0.103 M，則初始速率則下降到沒有鎂離子存在時的63 % ; 鎂離子濃度再增加到0.620 M，則初始速率則下降到沒有鎂離子存在時的48 %。 在pH值8-7的範圍內， pH值越小於8，則α-胰凝乳蛋白酶在水解速率上所受到的抑制也越大在，pH值為7.5時，初始速率則下降到pH值為8.0時的79 % ; pH值再下降到7.0，則初始速率則下降到pH值為8.0時的73 %。在遠紫外光-圓二色光譜的測定結果則顯示α-胰凝乳蛋白酶會因鎂離子濃度的增加而其二級結構也隨之變化，圖譜中顯示出α-螺旋的成份會隨著鎂離子濃度上升而漸漸增加 ; 遠紫外光-圓二色光譜的測定結果顯示在三級結構上並沒有太大的變化。螢光光譜測試中色胺酸螢光光譜、ANS螢光光譜、MIANS螢光光譜所顯示出來的結果都是，當鎂離子濃度逐漸增加時，其吸收的相對強度都隨之下降，表示當鎂離子濃度逐漸增加時，α-胰凝乳蛋白酶分子的構形較未含有鎂離子時的α-胰凝乳蛋白酶較為不同，其疏水性區域會漸漸縮小 ; 而pH值的改變對於α-胰凝乳蛋白酶的構形並沒有太大的影響。非色胺酸螢光光譜偵測結果則較沒有一個趨勢可循。

　　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+.

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