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研究生:林明煌
論文名稱:血紅素保存條件的探討
論文名稱(外文):Evaluation of Conditions for The Hemoglobin Storage
指導教授:宋信文
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:67
中文關鍵詞:血紅素變性血紅素血紅素保存
外文關鍵詞:hemoglobinmethemoglobinhemoglobin storage
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以血紅素為基材之人工代血可分為包覆型、基因重組型以及聚合型人工代血三種類型。我們實驗室所做的研究即是以血紅素為基材,經由適當的反應條件所製備出來的聚合型人工代血。而如果可以找出一個適合的條件來保存血紅素,則可省去每次由屠宰場取血再經由萃取得血紅素溶液的麻煩步驟。因此本研究的目的即為探討血紅素的保存條件。
血紅素在有氧的情況下會自動氧化成變性血紅素(metHb),變性血紅素無法與氧鍵結而失去攜氧功能,因此保存血紅素最大的目的就是抑制變性血紅素的生成。影響血紅素變性的因素包括了溫度、pH、血紅素濃度、溶液中的氧氣、superoxide與 H2O2 含量等等。而在低溫下以及適當的pH、濃度,並通入氣體以及添加superoxide dismutase(SOD)及catalase等添加劑將血紅素溶液中的氧氣、superoxide與H2O2去除,則可能可以抑制變性血紅素的生成。若是在冷凍狀態下則添加抗凍劑來減少冰晶所造成的傷害。
因此本研究分別就不同的溫度(4oC、-20 oC、-80 oC)、抗凍劑(glucose、sucrose)、pH(pH 6、pH 7.4、pH8.5)、濃度(6 g/dL、12 g/dL、20 g/dL)、通入不同氣體(N2、CO)及添加不同添加劑(SOD、catalase) 等,以保存6個月的時間來找出這些條件對保存血紅素的影響,並找尋較適當的保存條件。
實驗結果發現溫度升高會影響穩定血紅素的作用力而使得變性血紅素有上升的趨勢。而在冷凍的過程中加入glucose、sucrose等抗凍劑可以抑制變性血紅素的生成。
在4oC下pH 7.4及pH 8.5的血紅素變性值上升趨勢不快,而在pH 6保存條件下變性血紅素隨著時間的增加會呈現快速增加的現象。至於-20oC下pH的保存結果則與4oC相似,-80oC下因為能量更低,因此在pH 6下構形不會發生改變,不會有變性血紅素的生成,而glucose及sucrose能抑制變性血紅素的生成速度。
此外由實驗中發現冷凍-回溫過程會導致血紅素的沉澱析出,且冷凍-回溫次數會影響pH 6下血紅素的沉澱析出現象。實驗結果發現隨著冷凍-回溫次數的增加,血紅素的析出量有越來越多的趨勢,而添加sucrose可減少析出的現象,sucrose的濃度越高,則抑制析出的效果越好。且隨著冷凍-回溫次數的增加,血紅素變性值有上升的趨勢,而添加sucrose可有效抑制變性血紅素的生成速度。
因為我們保存的血紅素溶液中含有少量的不穩定的含氧血紅素(oxyHb),而不穩定的含氧血紅素間會利用cysteine之SH group形成雙硫鍵而產生自聚合分子,因此改變穩定的構形,而濃度越高時血紅素分子間的接觸機會越多,越容易有上述的情形發生。所以由濃度的實驗結果發現隨著濃度的增加,變性血紅素含量有越來越高的趨勢。
而由通氣體的實驗結果發現,通入氮氣並沒有將氧氣完全去除,溶液中仍存在少量的氧氣,可將血紅素氧化成變性血紅素,因此通入氮氣雖然有抑制變性血紅素生成的作用,效果卻不明顯。而一氧化碳與血紅素的結合能力為氧氣的250倍,通入一氧化碳不僅能將溶液中的氧氣去除,一氧化碳還會與氧氣競爭去和血紅素結合,且一氧化碳結合上去後不易釋放出來,可減少氧氣與血紅素的接觸機會。因此通入一氧化碳的效果明顯要比通入氮氣好很多。
由4oC下添加劑的實驗結果則發現SOD及catalase於4oC下被消耗掉後,無法將之後氧化過程中所產生的superoxide及H2O2去除以減少變性血紅素的生成。而catalase於-12oC至-75oC間失去活性,且SOD及 catalase並非抗凍劑,無法減少冷凍過程中冰晶形成及pH下降對血紅素所造成的傷害,因此在-20oC下添加SOD及 catalase無法抑制變性血紅素的生成速度。

內容 頁數
摘要----------------------------------------------------------------------------------I目錄------------------------------------------------------------------------------III
圖索引--------------------------------------------------------------------------V
表索引 VII
第一章 緒論
1.1 血紅素簡介 1
1.2 人工代血簡介 4
1.3 血紅素純化方式 7
1.4 蛋白質之四種結構 9
1.5 影響蛋白質穩定性之作用力 13
1.6 血紅素的保存條件 14
1.7 研究動機與目的 17
第二章 實驗方法
2.1 血紅素萃取純化 20
2.2 血紅素濃度測定 22
2.3 血紅素純度測定 23
2.4 變性血紅素含量測定 23
2.5 血紅素之保存條件探討 24
第三章 實驗結果與討論
3.1 血紅素純度測定結果 26
3.2 溫度對血紅素溶液保存結果的影響 28
3.3 抗凍劑對血紅素溶液保存結果的影響 30
3.4 pH對血紅素溶液保存結果的影響 33
3.5 濃度對血紅素溶液保存結果的影響 43
3.6 通氣體對血紅素溶液保存結果的影響 46
3.7 添加劑對血紅素溶液保存結果的影響 49
3.8 結論 51
參考文獻 53

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