Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22478
標題: 應用吳郭魚金屬硫蛋白和汞離子結合蛋白MerP於清除自由基之探討
Application of Tilapia metallothionein and MerP in free radical scavenging activity
作者: 吳文祥
Wu, Wen-Hsiang
關鍵字: metallothionein
金屬硫蛋白
Mercury-Ion-Binding Protein
free radical
汞離子結合蛋白
自由基
出版社: 生命科學系所
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摘要: 在生物系統中,Reactive oxygen species(ROS) (活性氧分子)藉由粒線體的電子傳遞鏈和NADP氧化酶的活化作用一直持續被製造產生。然而這些氧自由基在細胞體內卻會去破壞DNA、改變生化反應,損害細胞膜並且直接殺死細胞;以致這些氧自由基分子在癌症、心血管等疾病的形成上亦扮演著非常重要的角色。另一方面,Superoxide Dismutase(SOD) 、glutathione peroxidase (GPx) 、glutathione(GSH)和ascorbic acid這些抗氧化劑能夠藉由不同機轉去清除自由基,減低ROS所造成的傷害。Metallothionein(MT)是一個低分子量且富有cysteine的金屬結合蛋白,它能夠鏊合多種類的重金屬,而其中cysteine在則在清除自由基中扮演了最重要的角色。在水生環境裡,吳郭魚常被視為受污染水域之指標魚種而且其對重金屬耐受性比其他的魚類都好。正因如此,我們進行兩種不同自由基的體外試驗,比較吳郭魚和人類的MT清除自由基能力。在這項研究中,被純化的吳郭魚和人類的MT能夠有效地清除︰2,2-azinobis3- ethylbenzothiazoline- 6-sulfonic acid (ABTS•+) 和 1,1-diphenyl- picrylhydrazyl radical (DPPH• )等自由基,且其清除能力和MT濃度是呈現正相關的。雖然人與魚的MT皆遠高於glutathione的清除能力,但兩者的能力並無明顯的差別。另一方面,由重組大腸菌中純化出的陽性菌汞離子結合蛋白 (MerP)並且以化學合成革蘭氏陽性菌(G+ peptide:GMDCCPP)和革蘭氏陰性菌(G- peptide:GMTCAACP) MerP的金屬離子結合區域之胜肽,以DPPH•和ABTS•+進行自由基清除能力測試,同時以GSH作為此實驗的抗氧化劑對照組,在DPPH‧自由基抑制百分率((OD control-OD sample)/OD control x 100%))試驗中,MerP和GSH幾乎是相同的;對於ABTS•+自由基的部分,MerP的清除率則約多出對照組GSH多達20%。此實驗證明了汞離子結合蛋白MerP也能扮演一種抗氧化劑的角色。ABTS•+自由基清除試驗中,合成的胜肽和GSH具有同等的清除能力;而在DPPH‧自由基的部分,合成的胜肽抑制率則約多出對照組GSH超過30%。同時,當他們的半胱胺酸(cysteine)被丙氨酸(alanine)替換掉時即喪失清除的能力. 這些結果可以反應出細菌的重金屬結合蛋白質亦具有清除自由基的功能。
Reactive oxygen species (ROS) are continuously produced in biological system by the action of mitochondrial electron transport system and nicotinamide adenine dinucleotide phosphate (NADP) oxidase.These oxygen free radicals are cellular renegades and wreak havoc in biological system by damaging DNA,altering biochemical compounds,corroding cell membranes and killing cells outrightly.Such molecular mayhem plays a major role in the development of ailments like cancer,heart and lung diseases and cataracts.There are various key antioxidants such as superoxide dismutase (SOD),glutathione peroxidase (GPx),glutathione reductase (GR),catalase (CAT),glutathione (GSH) and ascorbic acid (AA) which mitigate the impact of ROS by different mechanisms.Antioxidant role for Metallothionein(MT) is well documented.MT is a low molecular weight and cysteine-rich metal-binding protein which could chelate many kinds of heavy metal and cysteine residues of MT are proposed to be the main contributors for its radical scavenging ability.In the water environment,tilapia shows higher tolerance than other kinds of fishes and was known as a biological marker for heavily polluted water areas.For these purposes,we compare the free radical scavenging activity using two different in vitro assays between Tilapia and Human metallothionein.In this study,the purified Tilapia and Huaman MT effectively scavenged the following free radicals: 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH• ).The radical scavenging effectwas found to be concentration-dependent.However,there is no differences on the inhibition ability between Tilapia and Human MT proteins.On the other hand, Bacillus mercuric ion binding protein (MerP) was purified from recombinant Escherichia coli host and oligopeptides that according to the metal binding motif of MerP occurred from either Gram-positive bacteria (G+ peptide: GMDCCPP) or Gram-negative bacteria (G- peptide: GMTCAACP) were synthesized.The DPPH• and ABTS•+ were used as free radicals while GSH was used as the control antioxidant.Though the free radicals scavenging ratio ((OD control-OD sample)/OD control x 100%)) in DPPH• assay was almost the same between MerP and GSH while MerP was 20% higher than GSH in ABTS•+ free radicals assay.The result suggest that MerP can play as an antioxidant.Furthermore,both of the synthesized oligopeptides performed scavenging ability as good as GSH in ABTS•+ assay and over 30% higher than GSH in DPPH• assay.Meanwhile,the scavenging abilities were lost while their cysteine residues were replaced by alanine residues.These results may reflect the duel functions of those heavy metals binding proteins.
URI: http://hdl.handle.net/11455/22478
其他識別: U0005-0202200812372300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0202200812372300
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