血紅蛋白結構變動與 溫度效應之關係 b2His 與 b6Glu 部位結構之
重要性
The Structure Basis of Temperature Effect on Hemoglobin: The Involvement of β2 His and β6 Glu
中文摘要
血紅蛋白是生物體內賴以維生的分子。過去的研究指出，其攜氧能力受到許多調 節因子調控而有所不同，溫度即為一重要因子。但對於溫度調控攜氧機能與血紅 蛋白構造在分子層面上之關係則尚未被解明，本研究擬以基因蛋白工程之方法來 探討。
本篇論文主要是求了解血紅蛋白中央空腔的 β2His 與 β6Glu 等位置構造對 血紅蛋白攜氧與溫度效應之關係而進行研究。先利用電腦完成血紅蛋白胺基酸序 列之比對與重組分子結構的模擬後，將中央空腔內上述之位置進行改變以引發分 子結構上的變動，作為實驗設計之分析確認。在實驗上利用 PCR mutagenesis 得 到其蛋白基因之變異株後，再轉殖進入特定蛋白表達系之大腸桿菌中大量製造出 重組血紅蛋白，並以一系列之管柱層析法純化並輔以可見光譜及電泳分析鑑定後 得到純度較高的重組血紅蛋白而進行功能及性能之測試。結果與先前所發現之天 然突變血紅蛋白相符合且重組血紅蛋白與人類血紅蛋白對於溫度的敏感性有差 異。r Hb (βH2E)與 r Hb (βH2E, E6K)之焓(DH)於酸鹼度 pH7.4 下，變化分別為 -5.39 及-2.47，即對於溫度的敏感度較人類血紅蛋白及重組野生株為低。肯定位 於血紅蛋白中央空腔之 β2 與 β6 位置對於溫度效應調控血紅蛋白之功能有 所關聯。
英文摘要
Hemoglobin (Hb) is a vital oxygen-transporting molecule in the aerobic organism.
Previous studies have shown that oxygen carrying function of Hb is regulated by various allosteric coffectors where temperature is an important one. However to date, the structure basis of enthalpy change in oxygen binding properties of Hb has not been elucidated. To investigate the positions in Hb that involved in the temperature effect, we have produced three site-directed mutant recombinant hemoglobins (rHbs):
r Hb (βH2E), r Hb (βH2E, E6K) and r Hb (βH2E, K82N, N108D) in this study.
Through a series of purification by chromatography, the obtained pure r Hbs have been subjected to oxygen binding function and stability analyses.
Results obtained from this study suggest that these residues: His β2 and Glu β6 in b globin indeed contribute to in the temperature effect of human adult hemoglobin (HbA) and consist with previous report by M. F. Perutz et al. that the electrostatic
state altered in the central cavity influences the Hb oxygen binding properties.
