為了探討雙硫鍵環狀小分子胜肽是否具有胰蛋白酶抑制劑的抑制效果。本研究以稻米芽鞘胰蛋白酶抑制劑含活性位置Domain III之胜肽片段來模擬向日葵花籽胰蛋白酶抑制劑(Sunflower Trypsin Inhibitor，SFTI)藉此測定其抑制之活性。
SFTI是一種很穩定的抑制劑，由14個胺基酸組成之環狀結構，具有一雙硫鍵結。本實驗以稻米芽鞘胰蛋白酶抑制劑去掉第91個位置的cysteine之Domain III，總共14個胺基酸序列，其序列為AFCNKMNPPTCRMD。使用簡單的氧化反應，讓整個結構利用雙硫鍵形成環狀，再去檢測抑制效果。從紅外線光光譜中，可知道環狀結構中有雙硫鍵的生成。且在指紋區600～620 cm-1處有看到雙硫鍵的stretching；在570～705 cm-1處有看到雙硫鍵的C－S的stretching，這些都可以判斷出，結構中有雙硫鍵的鍵結。
以N-α-benzoyl-L-arginine-p-nitroanilide (L-BAPNA)當受質對胰蛋白酶 (trypsin) 的Ki值為6.82 x 10-7 M；而直鏈結構的抑制劑其Ki值為8.65 x 10-7 M；此兩種結構的抑制劑皆是競爭型抑制。而此結果和稻米芽鞘胰蛋白酶抑制劑在相同條件下之Ki值4.00 x 10-7 M有些許差異；但和SFTI對β-trypsin抑制效果相比(Ki＝5.00 x 10-8 M)，其抑制能力較不好。但我們發現N-benzoyl-L-tryposine ethyl ester (BTEE) 為受質時的環狀抑制劑對胰凝乳蛋白酶(chymotrypsin) Ki值為3.08 x 10-8 M，而直鏈的抑制劑在相同條件下，其Ki值為8.06 x 10-8 M。抑制劑結果顯示出對胰凝乳蛋白酶之抑制效果較好。測量的Ki值結果與文獻上指出改變不同受質時，大部分胰蛋白酶抑制劑對此酵素的Ki範圍相符合(10-8～10-10 M)，說明此環狀抑制劑對chymotrypsin有好的抑制效果。

A cyclized peptide from sunflower seeds consists of 14 amino acids and a disulfide bond had been isolated and shown a promising activity as a serine protease inhibitor (SFTI). Therefore, in order to investigate whether a oligomer peptide with a disulfide bond possess potent protease inhibition activity, we have designed a 14-mer peptide, AFCNKMNPPTCRMD, based on the domiam III of rice coleoptile protease inhibitor (RTCI) from 79th amino acid to 92nd amino acid including the proposed active site of the 83rd lysine. This linear peptide was oxidized using known method, followed by purification by employing HPLC gave a compound, the IR spectrum of which showed there were extra absorptions between 600~620 cm-1(-S-S- stretching) and 570~705 cm-1 (-C-S- stretching) compared with that of linear peptide, indicating there was a disulfide bond. The linear and the disulfide bond cyclized peptides were assayed against trypsin and chymotrypsin. It was found that the Kis for cyclized and linear peptides against trypsin with L-BAPNA as substrate are 6.82 x 10-7 and 8.65 x 10-7, respectively. The inhibitory behavior of both showed they are competitive inhibitors. Compared with RTCI (Ki = 4.00 x 10-7) and SFTI (Ki = 5.00 x 10-8), these two peptides are not as active as both when against trypsin. However, when against chymotrypsin with BTEE as substrate, the Kis were 3.08 x 10-8 and 8.06 x 10-8 for cyclized and linear peptides, respectively. Compared with the reported activity for most protease inhibitors, the Ki of which were in the range of 10-810-10, our results showed that the disulfide bond cyclized peptide is a promising protease inhibitor. It is thus interesting to further study whether the proposed lysine residue is important in this peptide.

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