Naja naja atra chymotrypsin inhibitor (G-NNACI)是我們實驗室由Naja naja atra (台灣眼鏡蛇)的毒腺total RNA中所選殖出來的一個具有抑制凝乳蛋白活性的一個蛋白酶抑制劑。它由57個胺基酸組成，含有六個Cys形成三對雙硫鍵，序列比對結果認為它屬於Kunitz/BPTI family的蛋白酶抑制劑。由於BPTI的氮端胺基酸序列具有高度保留性，因此本實驗選擇在氮端以Ala-screening 定點突變，deletion及Domain swapping的方法試圖探討氮端序列在抑制劑蛋白酶活性上所扮演的角色，同時也探討氮端突變對於G-NNACI結構穩定所造成的影響。實驗結果顯示，所有的突變蛋白有相似的二級結構且都具有抑制凝乳蛋白的活性。但G-NNACI (R1A)、G-NNACI (P2A)及G-NNACI (△N3)的突變在鹼性環境下易發生雙硫鍵重組，在熱穩定及變性劑的變性試驗也呈現穩定性下降的情形。活性測試的結果發現在與凝乳蛋白反應三小時後，G-NNACI (R1A)、G-NNACI (P2A)及G-NNACI (△N3)的活性下降最明顯，故推論在這些氮端位置突變造成結構的不穩定進而影響其蛋白質的活性。先前研究指出台灣雨傘節前神經毒素

G-NNACI, a Naja naja atra chymotrypsin inhibitor consists of 57 amino acid residues cross-linked by three disulfide bridges and belongs to the Kunitz/BPTI superfamily, has been successfully cloned and expressed in our laboratory. Since snake venom non-neurotoxic Kunitz/BPTI inhibitors are most conserved in the core and in the N-terminal surface area, Ala-screening mutagenesis, deletion and Domain swapping on the N-terminus were carried out in this study to assess the role of N-terminus in G-NNACI. G-NNACI mutants with single amino acid substitution and deleted mutants were prepared. The secondary structure of all mutated proteins did not significantly alter as evidenced by CD spectra. Although all mutants are found to be functionally active as an inhibitor, their inhibitory potency against chymotrypsin differed. In contrast to G-NNACI and other mutants, R1A、P2A and △N3 mutants had a propensity to alter their disulfide linkages under basic conditions. The results of thermal and urea denaturation suggested that amino acid substitution and deletion at the N-terminus lead to a change in the structural stability of G-NNACI. Consequently, the inhibitory potency of G-NNACI mutants along with time was affected. B chain of

中文摘要••••••••••••••••••••••••1
英文摘要••••••••••••••••••••••••2
緒論••••••••••••••••••••••••••3
縮寫••••••••••••••••••••••••••9
實驗材料••••••••••••••••••••••••10
實驗方法••••••••••••••••••••••••12
實驗結果••••••••••••••••••••••••23
討論••••••••••••••••••••••••••31
圖表••••••••••••••••••••••••••36
參考文獻••••••••••••••••••••••••66

Antuch, W., Berndt, K. D., Chavez, M. A., Delfin, J. and Wuthrich, K. (1993) The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus. Eur J Biochem 212, 675-684.
Azim, M. K., Grossmann, J. G. and Zaidi, Z. H. (1999) Predicted three dimensional structural models of venom serine protease inhibitors and their interaction with trypsin and chymotrypsin. J Nat Toxin 8, 363-384.
Benishin, C. G. (1990) Potassium channel blockade by the B subunit of beta-bungarotoxin. Mol Pharmacol 38, 164-169.
Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F. Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. and Tasumi, M. (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 112, 535-542.
Bode, W. and Huber, R. (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur. J. Biochem 204, 433-451.
Cardle, L. and Dufton, M. J. (1997) Foci of amino acid residue conservation in the 3D structures of the Kunitz BPTI proteinase inhibitors: how do variants from snake venom differ? Protein Eng. 10, 131-136.
Chang, J. Y. and Ballatore, A. (2000) The structure of denatured bovine pancreatic trypsin inhibitor (BPTI). FEBS Lett 473, 183-187.
Chang, L. S., Chung, C., Huang, H. B. and Lin, S. (2001) Purification and characterization of a chymotrypsin inhibitor from the venom of Ophiophagus hannah (King Cobra). Biochem Biophys Res Commun 283:862-867.
Chen, C., Hsu, C. H., Su, N. Y., Lin, Y. C., Chiou, S. H. and Wu, S. H. (2001) Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus. J Biol Chem 276, 45079-45087.
Creighton, T. E. (1990) Protein folding. Biochem J 270, 1-16.
Eguchi, M., Kanbe, M. (1982) Changes in haemolymph protease inhibitors during metamorphosis of the silkworms, Bombyx mori L. (Lepidoptera: Bombycidae). Appl. Ent. Zool. 17, 179-187.
Eguchi, M. (1993) Protein protease inhibitors in insects and comparison with mammalian inhibitors. Comp. Biochem. Physiol. 105B, 449-456.
Favre, I., Moss, G. W., Goldenberg, D. P., Otlewski, J. and Moczydlowski, E. (2000) Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel. Biochemistry 39, 2001-2012.
Filippovich, I., Sorokina, N., Masci, P. P., de Jersey, J., Whitaker, A. N., Winzor DJ, Gaffney PJ and Lavin MF (2002) A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties. Br J Haematol 119, 376-384.
Gatehouse, A. M. R., Hilder, V. A., Boulter, D. (1991) Novel insect resistance using protease inhibitor genes. In: Dennis, E.S., Llewellyn, D.J. (Eds.), Plant Gene Research. Springer-Verlag, Wien, pp. 63-77.
Grzesiak, A., Krokoszynska, I., Krowarsch, D., Buczek, O., Dadlez, M. and Otlewski, J. (2000) Inhibition of six serine proteinases of the human coagulation system by mutants of bovine pancreatic trypsin inhibitor. J Biol Chem 275, 33346-33352.
Hagihara, Y., Shiraki, K., Nakamura, T., Uegaki, K., Takagi, M., Imanaka, T. and Yumoto, N. (2002) Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI). J Biol Chem 277, 51043–51048.
Hocman, G. (1992) Chemoprevention of cancer: protease inhibitors. Int J Biochem 24, 1365-1375.
James, A. E., Kraunsoe, Timothy, D. W., Claridge and Gordon, L. (1996) Inhibition of human leukocyte and porcine pancreatic elastase by homologues of bovine pancreatic trypsin inhibitor. Biochemistry 35, 9090-9096.
Jianwei, J., Meimin, Y. and Binggen, R. (2001) Construction and haracterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase. Biochimie 83, 1049-1055.
Judit, T. P., Irina, V. G., Clare, W. and George B. (2004) Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor. Biochemistry 43, 1591-1598.
Kang, S. H., Fuschs, M. S. (1980) Purification and partial characterization of a protease inhibitor from Drosophila melanogaster. Biochem. Biophys. Acta 611, 379-383.
Kondo, K., Toda, H., Narita, K. and Lee, C. Y. (1982) Amino acid sequence of beta 2-bungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains. J Biochem (Tokyo) 91, 1519-1530.
Kress, L. F. and Laskowski, M. Sr. (1967) The basic trypsin inhibitor of bovine pancreas. VII. Reduction with borohydride of disulfide bond linking half-cystine residues 14 and 38. J Biol Chem 242, 4925-4929.
Masci, P. P., Whitaker, A. N., Sparrow, L. G., de Jersey, J., Winzor, D. J., Watters, D. J., Lavin, M. F. and Gaffney, P. J. (2000) Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model. Blood Coagul Fibrinolysis 11, 385-393.
Moses, E. and Hinz, H. J. (1983) Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters. J Mol Biol 170, 765-776.
Murdock, L. L. and Shade, R.E. (2002) Lectins and protease inhibitors as plant defenses against insects. J Agric Food Chem 50, 6605-6611.
Perona, J. J., Tsu, C. A., Craik, C. S. and Fletterick, R. J. (1993) Crystal structures of rat anionic trypsin complexed with the protein inhibitors APPI and BPTI. J Mol Biol 230, 919-933.
Pritchard, L. and Dufton, M. J. (1999) Evolutionary trace analysis of the Kunitz/BPTI family of proteins: functional divergence may have been based on conformational adjustment. J Mol Biol 285, 1589-1607.
Ritonja, A., Meloun, B. and Gubensek, F. (1983) The primary structure of Vipera ammodytes venom trypsin inhibitor I. Biochim Biophys Acta 748, 429-35.
Ryan, C. A. (1990) Proteinase inhibitors in plants: genes for improvingdefenses against insects and pathogens. Ann. Rev. Phytopathol. 28, 425–449.
Smith, L. A., Lafaye, P. J., LaPenotiere, H. F., Spain, T. and Dolly, J. O. (1993) Cloning and functional expression of dendrotoxin K from black mamba, a K+ channel blocker. Biochemistry 32, 5692-5697.
Weissman, J. S. and Kim, P. S. (1991) Reexamination of the folding of BPTI: predominance of native intermediates. Science 253, 1386-1393.
Willmott, N., Gaffney, P. J., Masci, P. P. and Whitaker, A. N. (1995) Novel serine-protease inhibitor from the Australian brown snake, Pseudonaja textilis textilis: inhibition kinetics. Fibrinolysis 9, 1-8.
Wu, P. F., Wu, S. N., Chang, C. C. and Chang, L. S. (1998) Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait). Biochem J 334, 87-92.
Wu, P. F. and Chang, L. S. (2000) Genetic organization of A chain and B chain of beta-bungarotoxin from Taiwan banded krait (Bungarus multicinctus). A chain genes and B chain genes do not share a common origin. Eur J Biochem 267, 4668-4675.
Yousef, G. M., Elliott, M. B., Kopolovic, A. D., Serry, E. and Diamandis, E. P. (2004) Sequence and evolutionary analysis of the human trypsin subfamily of serine peptidases. Biochim Biophys Acta 1698, 77-86.
Zupunski, V., Kordis, D. and Gubensek, F. (2003) Adaptive evolution in the snake venom Kunitz/BPTI protein family. FEBS Lett 547, 131-136.