從無氧環境下生長的稻米芽鞘，經由陽離子交換管柱與膠體管柱層析的方式，我們分離出一分子量約在16kDa的胰蛋白脢抑制劑，發現此抑制劑為競爭型的抑制行為。為了探討此抑制劑的抑制行為，是否會受到環境上的影響，因此分別在不同的pH值與鎂離子下觀察。發現在pH7.8、pH8.8條件下，抑制劑與酵素有最大活性，在偏弱酸的條件下pH6.8活性較差。而離子濃度的大小，也會影響到抑制劑的抑制能力，實驗發現稻米芽鞘BBI在pH7.8，Mg2+濃度0M、0.5M、1.0M、1.5M、2.0M條件下，其Ki值分別為4.84×10-10M、4.56×10-10M、4.39×10-10M、3.07×10-10M、2.45×10-10M；在相同條件下，大豆BBI在pH7.8，Mg2+濃度0M、0.5M、1.0M、1.5M、2.0M條件下，其Ki值分別為4.21×10-11M、6.24×10-11M、8.46×10-11M、9.34×10-11M、1.18×10-10M。由CD光譜發現，不同pH值下加入抑制劑會改變酵素(trypsin)的二級結構，但是對於三級結構則沒有明顯的改變。二級結構的變化，也可以從Trp螢光得知，加入抑制劑後Trp螢光強度有明顯增加，表示加入BBI抑制劑，Trp殘基的環境有了改變；隨著鎂離子的加入螢光強度變得更強。ANS特性螢光可以作為探測蛋白質疏水性的測量，加入BBI抑制劑後，ANS螢光強度皆有增加的趨勢，即加入BBI抑制劑後，增加了Trypsin疏水性區域；隨著鎂離子的加入，ANS螢光強度度也變得較強，增加了Trypsin疏水性區域。螢光探針MIANS可用來判定半胱胺酸(Cysteine)殘基的數目與含量，發現加入抑制劑後，螢光強度皆有增強的現象，但是隨著鎂離子的加入螢光強度卻有降低的現象。

　A 16 kDa protease inhibitor had been purified from rice coleoptiles grown in hypoxia condition. This protease inhibitor showed a competitive inhibitor toward trypsin. In order to study the optimum inhibitory ability of this protease, we had measured its activity against trypsin under various pHs and in the presence of Mg2+. It was found that under weak basic condition, it showed better activity to inhibit trypsin. To further study the effect of the presence of Mg2+, various concentration of Mg2+ were added. It was found that at pH7.8 and with the presence of 0 M, 0.5 M, 1.0 M, 1.5 M, and 2.0 M of Mg2+, the respective Ki values were 4.84 x 10-10 M, 4.56 x 10-10 M, 4.39 x 10-10 M, 3.07 x 10-10 M, and 2.45 x 10-10 M, showing that the inhibitory activity in the presence of 2.0 M Mg2+ was about twofold of that without the presence of Mg2+. By contrast, same measurements for soybean protease inhibition activity, it was found that with the increase of the concentration of Mg2+ the inhibition activity decreased with the respective Ki values were 4.21 x 10-11 M, 6.24 x 10-11 M, 8.46 x 10-11 M, 9.34 x 10-11 M, and 1.18 x 10-10 M. It was found that the secondary structures were change with the presence of 2 M of Mg2+, whereas the tertiary structure was unchanged. The fluorescence study showed that the tryptophan fluorescence intensity increased indicating that there was a micro-environmental change around the tryptophan residues. ANS fluorescence also showed there was an increase for hydrophobic area in the presence of Mg2+. The MIANS fluorescence study showed that rice protease inhibitor resulted in the increase of intensity, however, with the presence of Mg2+, the intensity decreased.

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