摘要
太陽花籽胰蛋白酶抑制劑﹙sunflower trypsin inhibitor；SFTI﹚為一種很穩定的抑制劑，由14個胺基酸組成之環狀結構，且具有一雙硫鍵結。為了探討具有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，我們以實驗室所發現的稻米芽鞘胰蛋白酶抑制劑domain I去掉第八個位置的半胱胺酸﹙cysteine﹚具有33個胺基酸之片段，來探討抑制能力。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。測出其對胰蛋白酶﹙trypsin﹚，以L-BAPNA當受質的Ki值為7.97 x 10-7 M；而直鏈結構的抑制劑其Ki值為9.50 x 10-7 M；此兩種結構的抑制劑皆是競爭型抑制。而環狀抑制劑之抑制效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為減少約兩倍；而直鏈抑制劑和SFTI對β-trypsin抑制效果相比﹙Ki＝5.00 x 10-8 M﹚，抑制效果減少約19倍。而環狀抑制劑其Ki比SFTI差的原因為環狀結構抑制劑其構造上有一個長鏈所致，使得抑制能力較不好。但我們發現環狀的抑制劑對胰凝乳蛋白酶﹙chymotrypsin﹚，BPVANA當受質，其Ki值為1.47 x 10-9 M，以直鏈的抑制劑在相同條件下，其Ki值為1.35 x 10-8 M，與環狀的抑制劑相比減少了約10倍，發現抑制劑結構，會影響對胰凝乳蛋白酶之抑制效果。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胰凝乳蛋白酶有好的抑制效果。
從CD光譜發現，抑制劑直鏈結構α-helix的含量為2.4 %與環狀α-helix的含量3.0 %相比，變化沒有很大。而環狀結構在202 nm的位置有明顯的訊號，此為雙硫鍵所造成的訊號。此外，在210 nm的位置也有明顯的訊號，此為β-sheet的訊號，直鏈β-sheet的含量為54.44 %與環狀β-sheet的含量46.80 %。
從紅外線光譜中，發現環狀結構特性鋒和胱胺酸﹙cystine﹚相似，可知道其結構中，有雙硫鍵的生成。且在指紋區470～700 cm-1處有看到些許雙硫鍵的stretching，這些都可以判斷出，結構中有雙硫鍵的鍵結。
從拉曼光譜中，發現在1000 cm-1處有看到雙硫鍵之訊號，此處主要為使用633 nm波長激發時，胱胺酸所造成之雙硫鍵訊號，與文獻相符合。

Abstract
A 14 amino acid cyclic peptide with a disulfide bond protease inhibitor (SFTI) isolated from sunflower seeds showed a potential activity. It is thus interesting to explore the possibility for investigating small cyclic peptide having a disulfide bond as a potent protease inhibitor. We have isolated a protease inhibitor from rice coleoptiles with 133 amino acids, domain I of which has 33 amino acids. This peptide without the Cys7 was cyclized by a disulfide bond between Cys7 and Cys33, and was used as target for peptidomimetic study. It was found that the inhibitory activity in terms of Ki toward trypsin with L-BAPNA as substrate was 7.97 x 10-7 M and 9.50 x 10-7 M for disulfide bond cyclized peptide and non-cylized peptides, respectively.Both showed a competitive inhibitory behavior.The inhibitory constant (Ki) of this cyclo-target peptide was about twice of that for rice coleoptile protease inhibitor, Ki being 4.00 x 10-7 M and about 19 folds of that for SFTI, Ki being 5.00 x 10-8 M.Interestingly, it was found that the disulfide bond cyclized peptide and non-cyclized peptide showed a promising activity with Ki of 1.47 x 10-9 M and of 1.35 x 10-8, respectively, toward -chymotrypsin, which is competitive with the reported Ki value between 10-8 to 10-10 M,for various protease inhibitor toward -chymotrypsin, indicating this disulfide bond cyclized peptide showed a promising activity toward -chymotrypsin.
Circular Dichroism spectroscopic studies showed that the content of -helix for cyclo- and noncyclo-peptide were 2.4% and 3.0%, respective; whileas, the content of -sheet were 46.8% and 54.4%, respectively. The disulfide bond was characterized by Circular Dichroism showing a signal at 202 nm and by IR spectroscopy showing a disulfide bond stretching finger print absorption between 470 to 700 cm-1.

參考文獻
1. Gonzalo J, Dpmingo, Robin J. Leatherbarrow,Neil Freeman, Shila Patel,Malcolm Weir, "Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitor", Int. J. Peptide protein Res. , 46:79-87(1995)

2. David L., Ryan, C.A., "Around induced proteinase inhibitor in plant leaves: a possible defence mechanism against insects", Science(Washington) , 175:776-777(1972)

3. Yili Li, Qichen Huang, Shiwei Zhang, Shenping Liu, Chengwu Chi,and Youqi Tang, "Studies on an Artificial Trypsin Inhibitor Peptide Derived from the Mung Bean Trypsin Inhibitor:Chemical Synthesis, Refolding, and Crystallographic Analysis of It's complex with Trypsin", J. Biochem. , 116:18-25(1994)

4. David L., Michael M.Cox, Lehninger Principles of Biochemistry, Third Edition, Worth publishers, USA, pp 273-277, 626-627(2000)

5. Voet, Donald and Judith G. Voet, Biochemistry 2th , John Wiley, pp:518(1999)

6. Misao Tashiro, Kimikazu Hashino, Masake ShiozaKi, Fumio Ibuki, and Zensuke Maki, "The complete amino acid sequence of rice bran trypsin inhibitor", J. Biochem. , 102:297-306(1987)

7. Wen-Chi Hou, Hsien-Jung Chen and Yaw-Huei Lin, "Dioscorins from different Dioscorea species all exibit both carbonic anhydrase and trypsin inhibitor activities", Bot. Bull. Acad. Sin. , 41:191-196(2000)

8. Rosemarie W. Hammond, Donald E. Foard, and Brian A. Larkins, "Molecular Cloning and Analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean", The Journal of Biological Chemistry , 259(15):9883-9890(1984)

9. Randeep Rakwal, Ganesh Kumar Agrawal, Nam-Soo Jwa, "Characterization of a rice (Oryza sativa L.) Bowman-Birk proteinase inhibitor: tightly light regulated induction in response to cut,jasmonic acid, ethylene and protein phosphatase 2A inhibitor", Gene , 263:189-198(2001)

10. Yasuko Kato and Tsukasa Mastuda, " Glycation of Proteinous Inhibitors: Loss in Trypsin Inhibitory Activity by the Blocking of Arginine and Lysine Residues at Their Reactive Sites", J. Agric. Food Chem. , 45:3826-3831(1997)

11. Ping Chen, John Rose, Robert Love, Chin H. Wei, and Bi-Cheng Wang, " Reactive sites of an anticarcinogenic Bowman-Birk proteinase inhibitor are similar to other trypsin inhibitors", The Journal of Biological Chemistry , 267(3):1990-1994(1992)

12. Geoffrey L. Zubay, William W. Person, Dennis E. Vance, Principles of Biochemistry , New Wun Ching Developmental Publishing Co. Ltd. , 6-17, 7-11, 7-16(2002)

13. S. Luckett, R. Santiago Garcia, J. J. Barker1, Al. V. Konarev, P. R. Shewry, A. R. Clarke1 and R. L. Brady1, " High-Resolution Structure of a Potent, Cyclic Proteinase Inhibitor from Sunflower Seeds", J. Mol. Biol. , 290:525-533(1999)

14. Emmanuel Guiderdoni1, Maria Jos´e Cordero, Florence Vignols, Jos´e Manuel Garcia-Garrido, Magali Lescot1, Didier Tharreau1, Donaldo Meynard, Nicole Ferriere, Jean-Loup Notteghem and Michel Delseny, "Inducibility by pathogen attack and developmental regulation of the rice Ltp1 gene", Plant Molecular Biology , 49: 683–699(2002)

15. Ruth Arnon, Elchanan Maron, Michael Sela, and Chris Tlan B. Amfinnsen, "Antibodies Reactive with Native Lysozyme Elicited by a Completely Synthetic Antigen", Proc. Nat. Acad. Sci. , 68:1450-1455(1971)

16. James P. Tam, Cui-Rong Wu, Wen Liu, and Jing-Wen Zhang, "Disulfide Bond Formation in Peptides by Dimethyl Sulfoxide.Scope and Applications", J. Am. Chem. SOC. , 113:6657-6662(1991)

17. Xiaobing Tian and Eric Wickstrom, "Continuous Solid-Phase Synthesis and Disulfide Cyclization of Peptide-PNA-Peptide Chimeras", Org. Lett. , 4:4013-4016(2002)

18. W. Barry Edwards, Cynthia G. Fields, Carolyn J. Anderson, Tammy S. Pajeau, Michael J. Welch,t and Gregg B. Fields", J. Med. Chem. , 37:3749-3757(1994)

19. G. MezoÍ and F. Hudecz, "Synthesis and immunological studies of a-conotoxin chimera containing an immunodominant epitope from the 268±284 region of HSV gD protein", J. Peptide Res. , 55:7-17(2000)

20. 吳慧芬、呂麗琪，質譜儀分析技術的突破開展生化科技新領域，科學發展，362:49-51(2003)

21. 王文竹，透視生物巨分子結構，科學月刊，396:1033-1036(2002)

22. Daniel C. Liebler, "Introduction to Proteomics-Tools for the New Bioloogy", Humana Press Inc. , pp:55-76(2002)

23. Hennessey, J. P.; Johnson, W. C.: Jr. Information Content in the Circular Dichrosim of Proteins, Biochemistry , 20, pp:1085-1094(1981)

24. Adapted from Cornely K., Crespo E., Earley M., Kloter R., Levesque A., and Pickering M., Kinetics of Papain, An Introductory Biochemistry Laboratory Experiment, J. Chem. Ed. , 75(5):644-645(1999)

25. John E. Mole and H. Robert Horton Kinetics of apain-Catalyzed Hydrolysis ofa-N-Benzoyl-L-arginine-ρ-nitroanilide, Biochemistry , 12, No.5(1973)

26. 蔡育廷, "稻米芽鞘胰蛋白酶抑制劑抑制能力之研究", 國立成功大學化學系碩博士班論文2005年七月

27. Sheng Jiang, Peng Li, Sheau-Ling Lee, Cheng Yong Lin, Ya-Qiu Long, Michael D Johnson, Robert B Dickson, Peter P Roller, "Design and Synthesis of Redox Stable Analogues of Sunflower Trypsin Inhibitors (SFTI-1) on Solid Support, Potent Inhibitors of Matriptase", Org. Lett. , 9:9-12(2007)

28. John Wiley & Sons Ltd, Interpretation of Infrared Spectra, A Practical Approach , pp:10815–10837(2000)

29. WElLl QlAN and SAMUEL KRlM, " Vibrational Studies of the Disulfide Group in Proteins.Part V. Correlation of SS Stretch Frequencies with the CCSS Dihedral Angle in Known Protein Disulfide Bridges", Biopolymers , 32:321-326(1992)

30. Kenichi Oyaizu, Junya Katoh, Fumiaki Suzuki, Mitsutoshi Jikei, Kimihisa Yamamoto, Hiroyuki Nishide and Eishun Tsuchida, "Therrnolvsis of Reactive J Oligo(p-phenylene sulfide) Containing Disulfide Bond", Polymers for Advanced Technologies , Volume 2, pp:155-159(1991)

31. Yu-Zhi Su, Wen Dong, Jian-Hua Zhang, Jian-Hua Song, Yong-Hua Zhang, Ke-Cheng Gong, " Poly[bis(2-aminophenyloxy)disulfide]: A polyaniline derivative containing disulfide bonds as a cathode material for lithium battery", Polymer , 48:165-173(2007)

32. Harold E. Van Wart, Aaron Lewis, Harold A. Scheraga, and Frank D. Saeva, "Disulfide Bond Dihedral Angles from Raman Spectroscopy", Proc. Nat. Acad. Sci. , 70:2619-2623(1973)