鱧魚 (Channa maculata) 血紅蛋白之分子演化及功能適應性研究

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 HbIII ）。 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。

A Study on the Molecular Evolution and Functional Adaptation of

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 forillustrating the functional aspect of the structural evolutionin respiratory protein. To date, however, neither the structurenor the oxygen transport properties of this respiratoryhemoglobin has been L 嬙 rted yet. To gain insights into theallosteric nature of this fish hemoglobin, we have conducted aseries of structure and oxygen equilibrium studies. We haveisolated 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 tocharacterize the stability and dissociation rate of HbItetramer. An automatic recording oxygen equilibrium analyzer wasused to characterize the oxygen transport function ofhemoglobins. Hemoglobin of snakehead fish exhibits functionalcharacteristics in between the air breathing and the waterbreathing respiratory protein. Its oxygen transport is primarilyregulated by the cellular phosphates, especially ATP. Exhibitingno Root effect and a reduced alkaline Bohr effect. A partialprotein sequence corresponding the phosphate binding has beendetermined to confirm the structural basis this functionalcharacterize. On the other hand, the influence of proton on theoxygen transport function in this fish hemoglobin is temperaturedependent. Results obtained from this study provide a betterinsight into the benefit for the snakehead fish in adaptingbimodal breathing. Explaination of the structural basis of ATPas a strong effect is aided by a partial amino acid sequencedetermination analysis. The data provide an elueidation of howthe snakehead fish hemoglobin is designed to adapt the airbreathing at the molecular level.