在我研究中最主要探討台灣眼鏡蛇蛇毒蛋白中的磷酸二酯酶對於核苷酸的活性。我們使用了不同特性的管柱利用快速蛋白液相層析儀(FPLC)來純化出磷酸二酯酶來做實驗。在我的實驗中，我們發現了磷酸二酯酶是一個分子量約94.6 kDa的醣蛋白，由853胺基酸所組成的一個酵素蛋白，也在結構中發現兩顆鋅離子在活性區扮演重要的角色。在胺基酸序列比對中也發現與人類的ENPP1蛋白家族很相似，進而去探討相關的活性測試，想知道台灣眼鏡蛇毒中磷酸二酯酶扮演什麼角色。在研究中我們發現台灣眼鏡蛇蛇毒中的磷酸二酯酶對核苷酸種類活性有差異，因此我們由蛋白結構特性來解釋，且與出血性蛇種赤尾青竹絲的磷酸二酯酶對於核苷酸種類活性差異作比較，解釋了神經性蛇毒的台灣眼鏡蛇與出血性蛇毒的赤尾青竹絲作用機制差異，並且提出台灣眼鏡蛇磷酸二酯酶會使人體的免疫系統下降之假說。

Phosphodiesterase (PDE) from snake venom is a glycoprotein which digests extracellular nucleotides to affect the intercellular signaling (Fredholm et al., 2001) and the inhibition of ADP-induced platelet aggregation (Peng et al., 2011). In this study, we purified a PDE from Taiwan cobra venom (NA_PDE) by using gel filtration, ion exchange and affinity chromatography and its full length amino acid sequence (853 residues) was characterized from PDE-encoding cDNA. Sequence analysis reveals that NA_PDE belongs to Ectophosphodiesterase/nucleotide phosphohydrolase family (ENPP) (Greiner et al., 2013). Like ENPP, the purified NA_PDE hydrolyses ATP/ADP into AMP and PPi/Pi, GTP into GMP and PPi, CTP into CMP and PPi, UTP into UMP and PPi and also NAD into AMP and NMN. Compared to trimeresurus stejnegeri of Viperidae and ENPP1, NA_PDE showed a stronger enzymatic activity in hydrolysis of ATP. In addition, NA_PDE crystal structure resembles ENPP1 architecture and reveals a characteristic bimetallic (two zincs) active site and a nucleotide-binding pocket. These results provide the structural and functional insights of NA_PDE catalytic mechanism for digestion of nucleotides. Herewith we propose that NA_PDE might play a role in reducing immune system as it is known that ATP will stimulate the immune system (Akio & Michail, 2014).

Chapter 1 Introduction
1.1 Snake venom from Taiwan cobra (Naja naja atra) 3
1.2 Phosphodiesterase in Taiwan cobra venom 4
1.3 Role of nucleotides in human 9
1.4 Protein purification method: Chromatography 10
1.4.1 Gel Filtration Chromatography 10
1.4.2 Ion Exchange Chromatography 10
1.4.3 Affinity Chromatography 11
1.5 Enzyme kinetics 11
Chapter 2 Materials and Methods
2.1 Materials 13
2.2 Purification of the phosphodiesterase from Taiwan cobra (Naja naja atra) 13
venom
2.3 Phosphodiesterase Crystallization & Co-Crystallization 15
2.4 Data collection and structure determination of phosphodiesterase 15
2.5 Alkaline phosphatase activity assay 16
2.6 Phosphodiesterase inhibition assay with EDTA 16
2.7 Phosphodiesterase activity assay 16
2.8 Circular Dichroism studies on phosphodiesterase protein 17
2.9 Determination of extinction coefficient at 280 nm 17
2.10 Isothermal titration calorimetry measurements 17



Chapter 3 Results
3.1 Purification of the PDE from Taiwan cobra 18
3.2 Phosphodiesterase secondary structure identification 19
3.3 PDE crystal and data collection 19
3.4 Extinction coefficient of PDE at 280nm 19
3.5 PDE activity with specific substrates 20
3.6 Isothermal titration calorimetry measurements 21
Chapter 4 Discussion 35
Conclusion 38
Reference 47
Appendix 55

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