新抑癌素 (Neocarzinostatin)是一個天然的強力抗腫瘤抗生素。它由一個酸性蛋白，以及蛋白內具有藥物活性的烯雙炔生色團 (enediyne chromophore)所組成。硫醇能引發烯雙炔的環化作用，而蛋白除了保護極不穩定的生色團，更具有引導生色團走不同環化路徑的功能。然而為何蛋白擁有這種神奇的功能，則是個很值得探索的問題。氫鍵、疏水性的交互作用力等，皆曾被學者認為是可能的原因。本研究探討可能因素為蛋白的雙硫鍵，看其對生色團環化作用是否有影響。而選擇雙硫鍵是因其為維繫蛋白三級結構主要力量之一，且新抑癌素蛋白含有兩個雙硫鍵，分別是Cys37-Cys47及Cys88-Cys93，其中Cys37-Cys47正好位於穴口內部烯雙炔生色團的下方，因此很值得我們去探討雙硫鍵對生色團環化作用的影響。
實驗採用分生技術來定點突變蛋白的胺基酸，主要是以絲胺酸 (serine)來置換掉半胱胺酸 (cysteine)，藉以消除雙硫鍵，我們首先選擇位於生色團附近之雙硫鍵 (Cys37-Cys47)，接著修飾Cys88-Cys93。我們將建構好的質體轉殖進入大腸桿菌以表達修飾的蛋白，接著進行純化，然後用質譜儀確認蛋白雙硫鍵是否已斷開，再用圓二色光譜儀 (Circular Dichroism Spectroscopy)檢測蛋白結構的一致性。然後將蛋白與生色團重新結合進行環化反應，以高效率液相層析儀鑑定生色團環化路徑有無改變，比較其內之生色團環化反應與原來的蛋白有何差異，以更進一步地去分析出生色團環化反應路徑。在蛋白方面，經由純化之後得到很純且分子量正確的apoNCS和突變C93S蛋白，而突變C37S蛋白雖經純化之後得到很純的蛋白，但其分子量不正確，所以只針對apoNCS和突變C93S蛋白來做比較，將蛋白與生色團重新結合之後，進行蛋白與生色團鍵結穩定性的探討及生色團環化反應的比較，由實驗的結果顯示突變C93S蛋白的鍵結穩定性並沒有apoNCS好及生色團環化反應之後的產物比例並沒有明顯的改變，可能是Cys88-Cys93這條雙硫鍵對生色團環化反應的影響不是主要的，應是離穴口較近的Cys37-Cys47雙硫鍵對生色團環化反應的影響會比較大些，但需得到突變C37S蛋白或突變C47S蛋白之後，才能去驗證這個假設。

目 錄 頁碼
圖目錄 5
表目錄 9
英文摘要 10
中文摘要 13
第一章 緒論 15
第二章 材料與方法 27
2-1 製備突變質體 27
突變引子 27
PCR製備突變質體 27
2-2 勝任細胞 (competent cell)的配製 28
2-3 突變質體的transformation 29
2-4 突變質體的萃取 30
2-5 DNA電泳分析 31
2-6 DNA定序 32
2-7 蛋白的表現與純化 32
2-8 SDS-PAGE分析 37
2-9 蛋白的HPLC分析 37
2-10 蛋白的質譜分析 39
2-11 蛋白的CD光譜分析 39
2-12 生色團的萃取及定量 40
2-13 生色團與蛋白的再結合 42
2-14 生色團環化反應分析 43
第三章 結果 45
3-1 蛋白胺基酸的突變 45
3-2 蛋白的表現與純化 46
3-3 蛋白與生色團鍵結穩定性的探討 48
3-4 蛋白雙硫鍵影響生色團環化反應的比較 49
第四章 討論 51
第五章 總結 53
第六章 參考文獻 54
圖目錄 頁碼
Figure 1、Structure of Holo-NCS 16
Figure 2、Primary structure of NCS apoprotein 17
Figure 3、Structure of NCS chromophore 19
Figure 4、Except NCS chromophore, other antitumor drugs
included enediyne from nature. 20
Figure 5、UV spectrum of product 1 and 2 23
Figure 6、Structure of pCAL-n-EK vector 26
Figure 7、1％ DNA gel of mutant C37S plasmid DNA 59
Figure 8、The sequence of mutant C37S compare with ApoNCS 60
Figure 9、1％ DNA gel of mutant C93S plasmid DNA 61
Figure 10、The sequence of mutant C93S compare with ApoNCS 62
Figure 11、1％ DNA gel of mutant C93S plasmid DNA 63
Figure 12、The sequence of mutant C93S compare with ApoNCS 64
Figure 13、15％ SDS-PAGE analysis of uninduced and induced protein 65
Figure 14、15％ SDS-PAGE analysis of mutant C37S protein 66
Figure 15、15％ SDS-PAGE analysis of mutant C93S protein 67
Figure 16、15％ SDS-PAGE analysis of ApoNCS 68
Figure 17、15％ SDS-PAGE analysis of mutant C37S protein 69
Figure 18、15％ SDS-PAGE analysis of mutant C93S protein 70
Figure 19、DEAE elution profile of ApoNCS 71
Figure 20、DEAE elution profile of mutant C37S protein 72
Figure 21、DEAE elution profile of mutant C93S protein 73
Figure 22、15％ SDS-PAGE analysis of ApoNCS 74
Figure 23、15％ SDS-PAGE analysis of mutant C37S protein 75
Figure 24、15％ SDS-PAGE analysis of mutant C93S protein 76
Figure 25、UV spectrum of ApoNCS 77
Figure 26、UV spectrum of mutant C37S protein 78
Figure 27、UV spectrum of mutant C93S protein 79
Figure 28、HPLC analysis of mutant C37S protein 80
Figure 29、HPLC analysis of mutant C93S protein 81
Figure 30、MS spectrum of ApoNCS 82
Figure 31、MS spectrum of mutant C37S protein 83
Figure 32、MS spectrum of mutant C93S protein 84
Figure 33、CD spectrum of ApoNCS 85
Figure 34、CD spectrum of mutant C37S protein 86
Figure 35、CD spectrum of mutant C93S protein 87
Figure 36、Comparsion of protein CD spectrum 88
Figure 37、CD spectrum of HoloNCS standard 89
Figure 38、CD spectrum of reconstituted holoNCS of ApoNCS 90
Figure 39、CD spectrum of reconstituted holoNCS of mutant C93S protein 91
Figure 40、Comparsion of holoNCS CD spectrum 92
Figure 41、HPLC analysis of extracted chromophore 93
from holoNCS standard
Figure 42、HPLC analysis of reconstituted holoNCS 94
of ApoNCS
Figure 43、HPLC analysis of reconstituted holoNCS 95
of mutant C93S protein
Figure 44、HPLC analysis of reconstituted holoNCS 96
of mutant C93S protein reacted with Glutathion
Figure 45、HPLC analysis of reconstituted holoNCS of mutant C37S protein97
Figure 46、HPLC analysis of reconstituted holoNCS 98
of mutant C37S protein reacted with Glutathione
Figure 47、HPLC analysis of reconstituted holoNCS 99
of ApoNCS reacted 5 hours at 0℃,
pH 7.4 Tris-HCl buffer.
Figure 48、HPLC analysis of reconstituted holoNCS 100
of mutant C93S protein reacted 5 hours at 0℃,
pH 7.4 Tris-HCl buffer.
Figure 49、HPLC analysis of reconstituted holoNCS 101
of ApoNCS reacted 24 hours at 0℃,
pH 7.4 Tris-HCl buffer.
Figure 50、HPLC analysis of reconstituted holoNCS 102
of mutant C93S protein reacted 24 hours at 0℃,
pH 7.4 Tris-HCl buffer.
Figure 51、HPLC analysis of reconstituted holoNCS 103
of ApoNCS reacted with Ethyl 2-mercaptoacetate
at 0℃ 5 hours.
Figure 52、HPLC analysis of reconstituted holoNCS 104
of mutant C93S protein reacted with
Ethyl 2-mercaptoacetate at 0℃ 5 hours.
Figure 53、HPLC analysis of holoNCS standard 105
reacted with 3-mercapto-1,2-propanediol
Figure 54、HPLC analysis of holoNCS standard 106
reacted with 2-mercaptoethanol.
Figure 55、HPLC analysis of reconstituted holoNCS
of ApoNCS reacted with 3-mercapto-1,2-propanediol. 107
Figure 56、HPLC analysis of reconstituted holoNCS 108
of ApoNCS reacted with 2-mercaptoethanol.
Figure 57、HPLC analysis of reconstituted holoNCS 109
of mutant C93S protein
reacted with 2-mercaptoethanol.
表目錄 頁碼
Table 1 、Reaction of reconstituted holoNCS and thiol 110
Table 2、Binding stability comparison of reconstituted holoNCS 111
Table 3、Comparison of ratio of product 1 and 2 of reconstituted holoNCS reacted with Ethyl 2-mercaptoacetate at 0℃ 5 hours 111
Scheme 1、Cycloaromatization pathway of NCS chromophore. 22

1. Ishida, N., Miyazaki, K., Kumagai, K., and Rikimaru, M. “Neocarzinostatin, an antibiotic of high molecular weight.” J. Antibiotics 1965, 18, 68-76.
2. Napier, M. A., Holmquist, B., Strydom, D. J., and Goldberg, I. H. “Neocarzinostatin: spectral characterization and separation of a non-protein chromophore.” Biochem. Biophys. Res. Commun. 1979, 89, 635-642.
3. Koide, Y., Ishii, F., Hasuda, K., Koyama, Y., Katamine, S., Kitame, F., and Ishida, N. “Isolation of a non-protein component from neocarzinostatin and their biological activities.” J. Antibiotics 1980, 33, 342-346.
4. Kappen, L. S., Napier, M. A., and Goldberg, I. H. “Roles of chromophore and apo-protein in neocarzinostatin action.” Proc. Natl. Acad. Sci. 1980, 77, 1970-1974.
5. Edo, K., Mizugaki, M., Koide, Y., Seto, H., Furihata, K., Oake, N., and Ishida, N. “The struture of neocarzinostatin chromophore possessing a novel bicyclo-[7,3,0] dodecadiyne system.” Tetrahedron Lett. 1985, 26, 331-334.
6. Goldberg, I. H. “Mechanism of neocarzinostatin action: Role of DNA microstructure in determination of chemistry of bistranded oxidative damage.” Acc. Chem. Res. 1991, 2, 191-198.
7. Meienhofer, J., Maeda, H., Glaser, C. B., Czombos, J., and Kuromizu, K. “Primary structure of neocarzinostatin, an antitumor protein.” Science 1972, 178, 875-876.
8. Maeda, H.; Glaser, C. B.; Kuromizu, K.; Meienhofer, J. “Structure of the antitumor protein neocarzinostatin. Amino acid sequence.” Arch. Biochem. Biophys. 1974, 164, 379-385.
9. Gibson, B. W., Herlihy, W. C., Samy, T. S. A., Hahm, K. S., Maeda, H., Meienhofer, J., and Biemann, K. A. “A revised primary structure for neocarzinostatin based on fast atom bombardment and gas chromatographic-mass spectrometry.” J. Biol. Chem. 1984, 259, 10801-10806.
10. Kuromizo, K., Tsunasawa, S., Maeda, H., Abe, O., and Sakiyama, F. “Reexamination of the primary structure of an antitumor protein, neocarzinostatin.” Arch. Biochem. Biophys. 1986, 246, 199-205.
11. Sakata, N., Minamitani, S., Kanbe, T., Hori, M., Hamada, M., and Edo, K. “The amino acid sequence of neocarzinostatin apoprotein deduced from the base sequence of the gene.” Biol. Phar. Bull. 1993, 16, 26-28.
12. Koide, Y., Ito, A., Ishii, F., Koyama, Y., Edo, K., and Ishida, N. “Reconstitution of neocarzinostatin.” J. Antibiotics 1982, 35, 766-769.
13. Takashima, H., Amiya, S., and Kobayashi, Y. “Neocarzinostatin: interaction between the antitumor-active chromophore and the carrier protein.” J. Biochem. (Tokyo) 1991, 109, 807-810.
14. Kim, K. H., Kwon, B. M., Myers, A. G., and Rees, D. C. “Crystal structure of neocarzinostatin, an antitumor protein-chromophore complex.” Science 1993, 262, 1042-1046.
15. Kuromizo, K., Abe, O., and Maeda, H. “Location of the disulfide bonds in the antitumor protein neocarzinostatin.” Arch. Biochem. Biophys. 1991, 286, 569-673.
16. Teplyakov, A., Obmolova, G., Wilson, K., and Kuromizu, K. “Crystal struture of apo-neocarzinostatin at 0.15-nm resolution.” Eur. J. Biochem. 1993, 213, 737-741.
17. Albers-Schonberg, G., Dewey, R. S., Hensens O. D., Liesch, J. M., Napier, M. A., and Goldberg, I. H. “Neocarzinostatin: chemical characterization and partial structure of the non-protein chromophore.” Biochem. Biophys. Res. Coummun. 1980, 95, 1351-1356.
18. Schreiber, S. L., and Kiessling, L. L. “Synthesis of the bicyclic core of the esperamicin/calichemicin class of antitumor agents.” J. Am. Chem. Soc. 1988, 110, 631-633.
19. Napier, M. A., Goldberg, I. H., Hensens, O. D., Dewey, R. S., and Liesch, J. M. “Neocarzinostatin chromophore: presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms.” Biochem. Biophys. Res. Commun. 1981, 100, 1703-1712.
20. Kappen, L. S., Napier, M. A., Goldberg, I. H., “Roles of chromophore and apo-protein in neocarzinostatin action.” Proc. Natl. Acad. Sci. 1980, 77, 1970-1974.
21. Povirk, L.F., Dattagupta, N., Warf, B.C., Goldberg, I. H. “Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation.” Biochemistry, 1981, 14, 4007-4014
22. Schroeder, D. R., Colson, K. L., Klohr, S. E., Zein, N., and Langley, D. R. “Isolation ; Structure Determination ; and Proposed Mechanism of Action for Artifacts of Maduropeptin Chromophore.” J. Am. Chem. Soc. 1994, 116, 9351-9352.
23. Leet, J. E., Schroeder, D. R., Hofstead, S. J., Golik, J., and Colson, K. L. “Kedarcidin; a New Chromoprotein Antitumor Antibiotic: Structure Elucidation of Kedarcidin Chromophore.” J. Am. Chem. Soc. 1992, 114, 7946-7948.
24. Yoshida, K., Minami, Y., Azuma, R., Saeki, M., and Otani, T. “Structure and Cycloaromatization of a Novel Enediyne; C-1027 Chromophore.” Tetrahedron Lett. 1993, 34, 2637-2640.
25. Ando, T., Ishii, M., Kajiura, T., Kameyama, T., and Miwa, K. “A new non-protein enediyne antibiotic N1999A2: Unique enediyne chromophore similar to neocarzinostatin and DNA cleavage feature.” Tetrahedron Lett. 1998, 39, 6495-6498.
26. Golik, J., Dubay, G., Groenewold, G., Kawaguchi, H., and Konishi, M. “Esperamicins; a Novel Class of Potent Antitumor Antibiotics. Structures of Esperamicins-A1, Esperamicin-A2, and Esperamicin-A1b.” J. Am. Chem. Soc. 1987, 109, 3462-3464.
27. Lee, M. D., Dunne, T. S., Chang, C. C., Ellestad, G. A., and Siegel, M. M. “Calichemicins; a Novel Family of Antitumor Antibiotics. Chemistry and Structure of Calichemicin-Gamma-1.” J. Am. Chem. Soc. 1987, 109, 3466-3468.
28. Konishi, M., Ohkuma, H., Matsumoto, K., Tsuno, T., and Kamei, H. “Dynemicin; a Novel Antibiotic with the Anthraquinone and 1, 5-Diyn-3-Ene Subunit.” J. Antibiotics 1989, 42, 1449-1452.
29. McDonald, L. A., Capson, T. L., Krishnamurthy, G., Ding, W. D., and Ellestad, G. A. “Namenamicin; a new enediyne antitumor antibiotic from the marine ascidian Polysyncraton lithostrotum.” J. Am. Chem. Soc. 1996, 118, 10898-10899.
30. Oku, N., Matsunaga, S., and Fusetani, N. “Shishijimicins A-C, novel enediyne antitumor antibiotics from the ascidian Didemnum proliferum.” J. Am. Chem. Soc. 2003, 125, 2044-2045.
31. Myers, A. G. “Proposed structure of the neocarzinostatin chromophore- methyl thioglycolate adduct; a mechanism for the nucleophilic activation of neocarzinostatin.” Tetrahedron Lett. 1987, 28, 4493-4496.
32. Myers, A. G., Cohen, S. B., and Kwon, B. M. “DNA cleavage by neocarzinostatin chromophore. Establishing the intermediacy of chromophore-derived cumulene and biradical species and their role in sequence-specific cleavage.” J. Am. Chem. Soc. 1994, 116, 1670-1682.
33. Kappen, L. S., Goldberg, I. H., and Liesch, J. M. “Identification of Thymidine-5''-Aldehyde at DNA Strand Breaks Induced by Neocarzinostatin Chromophore.” Proc. Natl. Acad. Sci. 1982, 79, 744-748.
34. Jung, G., and Kohnlein, W. “Neocarzinostatin: controlled release of chromophore and its interaction with DNA.” Biochem. Biophys. Res. Commun. 1981, 89, 176-183.
35. Kappen, L. S., and Goldberg, I. H. “Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells.” Biochemistry 1980, 19, 4786-4790.
36. Hirama, M., and Tanaka, T. “Molecular recognition in neocarzinostatin complex: how does the apoprotein bind specifically and stabilize the chromophore?” Pure Appl. Chem. 1994, 66, 791-796.
37. Chin, D. H. “Rejection by protein through charges rather than sizes.” Chem. Eur. J. 1999, 5, 1084-1090.
38. Sugiyama, H., Yamashita, K., Nishi, M., and Saito, I. “A novel cyclization pathway of neocarzinostatin chromophore by thiol under physiological conditions.” Tetrahedron Lett. 1992, 33, 515-518.
39. Sugiyama, H., Yamshita, K., Fujiwara, T., and Saito, I. “Apoprotein-Assisted Unusual Cyclization of Neocarzinostatin Chromophore.” Tetrahedron 1994, 50, 1311-1325.
40. Myers, A. G., Stephen, P. A., and Robert, W. L. “A new and unusual pathway for the reaction of neocarzinostatin chromophore with thiols. Revised structure of the protein-directed thiol adduct.” J. Am. Chem. Soc. 1996, 118, 4725-4726.
41. 尚未發表數據
42. 尚未發表數據
43. Chin, D.-H., and Tseng, M.-C. “Abnormal cycloaromatization of Neocarzinostatin induced by sugar thiols.” Tetrahedron Lett. 1997, 38, 2891-2894.
44. 尚未發表數據
45. Wyborski, D. L., Bauer, J. C., Zheng, C., Felts, K., and Vaillancourt, P. “An Escherichia coli expression vector that allows recovery of proteins with native n-termini from purified calmodulin-binding peptide fusions.” Protein Expr. Purif. 1999, 16, 1-10.
46. Chin, D.-H., Tseng, M. C., Chuang, T. C., and Hong, M. C. “Chromatographic and spectroscopic of thiol induced cycloaromatizations of enediyne in neocarzinostatin.” Biochim. Biophys. Acta. 1997, 1336, 43-50.