鱧魚(Channa maculata)血紅蛋白之分子演化及功能適應性研究 A Study on the Molecular Evolution and Functional Adaptation of
中文摘要
鱧魚學名 Channa maculata，其呼吸器官除了鰓能直接吸取水中微量 溶氧（約 3％）外，另已演化出副呼吸器官以便直接呼吸空氣（含氧約 21 ％）。因此鱧魚的血紅蛋白在以功能的觀點來闡明蛋白在結構演化上 的分子基礎提供一個很好的例子。而且對於生理上進化出此類能呼吸空氣 之副呼吸器官的魚類，其血紅蛋白為何能適應水中及陸上的呼吸能力尚未 被探討過，所以本篇論文就此鱧魚血紅蛋白的結構及功能特性作研究，並 由此蛋白的結構特性來探討其分子之演化。 為了瞭解鱧魚的血紅蛋白 的功能及結構特性，我們做了一系列的色層分析及氧合平衡研究。 首先
，用離子交換樹脂層析法分離全血溶物（ whole trait ），得一主要血 紅蛋白組成（ Hb I ）及三種微量的血紅蛋白組成（ HbIx，Hb II 及 Hb III ）。 膠質層析法的數據指出 Hb I 具有四聚體低解離的特性。分析 其氧合平衡性質得知鱧魚血紅蛋白已具有部份陸上動物血紅蛋白的特性，
其氧氣輸送機能受細胞內 ATP 之調控，其氫離子之影響能隨溫而變，有 益於鱧魚之兩棲呼吸。這些功能特性之結構基礎可由其部份氨基酸序列來 解釋。 為了知曉鱧魚血紅蛋白之球蛋白（ globin ）的氨基酸序列，
所以我們從求得其cDNA 序列著手。 由鱧魚全血中抽取細胞總和 RNA，經
反轉錄反應及聚合鏈鎖反應已經可得 ?蛋白部份 DNA 序列。 論文
中之分析的結果提供了鱧魚血紅蛋白適應兩棲呼吸在分子層面上如何演變 及修飾之說明。
英文摘要
Snakehead fish (Channa maculata) is a bimodal breathing fish, able to acquire oxygen from water as well from air directly.
Hemoglobin of this fish may therefore be an excellent model for illustrating the functional aspect of the structural evolution in respiratory protein. To date, however, neither the structure nor the oxygen transport properties of this respiratory
hemoglobin has been L 嬙 rted yet. To gain insights into the allosteric nature of this fish hemoglobin, we have conducted a series of structure and oxygen equilibrium studies. We have isolated a major hemoglobin (HbI) and three minor hemoglobins (HbIx, HbII, and HbIII)from the whole trait using an ion-
exchange chromatography. A gel chromatography was employed to characterize the stability and dissociation rate of HbI
tetramer. An automatic recording oxygen equilibrium analyzer was used to characterize the oxygen transport function of
hemoglobins. Hemoglobin of snakehead fish exhibits functional characteristics in between the air breathing and the water
breathing respiratory protein. Its oxygen transport is primarily regulated by the cellular phosphates, especially ATP. Exhibiting no Root effect and a reduced alkaline Bohr effect. A partial protein sequence corresponding the phosphate binding has been determined to confirm the structural basis this functional
characterize. On the other hand, the influence of proton on the oxygen transport function in this fish hemoglobin is temperature dependent. Results obtained from this study provide a better insight into the benefit for the snakehead fish in adapting bimodal breathing. Explaination of the structural basis of ATP as a strong effect is aided by a partial amino acid sequence determination analysis. The data provide an elueidation of how the snakehead fish hemoglobin is designed to adapt the air breathing at the molecular level.
