鴨眼球蛋白之酵素研究

Abstract

眼球蛋白（crystallin）原本被認為只是－種結構性蛋白，藉其以維持眼球的透明度（transparency）及光透明（optical clarity)。 最近，發現眼球蛋白與－些具有代謝功能的酵素存在有相似的胺基酸序列或相類似的酵素活性。而在鴨的眼球內含有兩種眼球蛋白具有很強的酵素活性: （1）ε-眼球蛋白具有乳酸去氫酵素活性（LDH activity) （2）δ-眼球蛋白具有精氨琥珀酸裂解酵素活性（ASase activity)。 本篇論文主要分為三個部份來討論： ( A ）鴨眼球蛋白的製備與純化 從鴨頭取下鴨眼球，除去眼球外膜並均質化與離心後取上清液（此上清液即是粗眼球蛋白液）。利用凝膠層析法只可由粗眼球蛋白中部分分離出ε-眼球蛋白及δ-眼球蛋白，而利用離子交換層析法則可以由粗眼球蛋白中分離出純度相當高的ε-眼球蛋白，但卻只能由粗眼球蛋白中部分純化出δ-眼球蛋白。純化後的ε-眼球蛋白分別利用聚凝膠電泳法與高效能液相層析法來鑑定其純度 ( B ) ε-眼球蛋白酵素活性之研究 首先測定ε-眼球蛋白之酵素活性對熱的穩定度，發現不論是未純化或是純化後的ε-眼球蛋白之酵素活性於25℃下，21天後仍具有百分之60以上的酵素活性，而在62℃下其酵素活性可以維持8小時以上，顯示ε-眼球蛋白之酵素活性是相當穩定的。然後利用各種不同的2-酮基酸為ε-眼球蛋白的受質進行酵素催化反應，向ε-眼球蛋白對這些2-酮基酸之酵素反應性結果來推測ε-眼球蛋白之受質結合部位（substrate binding site）的性質，結果顯示ε-眼球蛋白之受質結合部位與其他來源之乳酸去氫酵素有相當顯著的差異。向於ε-眼球蛋白具有相當穩定的酵素活性，故可以利用ε-眼球蛋白為生物有機催化劑將2-酮基丁酸及2-酮基戊酸分別還原成具光學活性的2-酮基丁酸及2-羥基戊酸，而2-羥基丁酸及2-羥基戊酸分別是合成昆蟲性荷爾蒙與殺黴菌劑的先驅物。結果顯示ε-眼球蛋白是相當適合做為生物有機催化劑合成重要的有機化合物。 (C)眼球蛋白酵素活性之研究 首先測定δ-眼球蛋白之酵素活性對受質類似物（substrate analogue）之反應性，以推測δ-眼球蛋白之受質結合部位的性質，結果顯示δ-眼球蛋白對其受質的接受度有相當程度的限制，只能接受少數幾個受質。此外，由於δ-眼球蛋白具有相當穩定的酵素活性，故可以利用δ-眼球蛋白為生物有機催化劑將L-精胺酸與反丁烯二酸合成為L-精氨琥珀酸，而L-精氨琥珀酸可以用來檢定精氨琥珀酸裂解酵素之活性，藉以診斷是否有精氨琥珀酸尿症的疾病。結果顯示δ-眼球蛋白可以很有效的催化合成L-精氨琥鉑酸，並且δ-眼球蛋白可以重複的使用，但其缺點是所能反應的受質只有少數幾個，故未能催化合成出多種產物。

Previously, crystallins were thought to play only a structural role in the maintenance of lens transparency and optical clarity. Recent development in thecrystallin chemistry of various animal eye lens has revealed the salient features of sequence similarities between crystallins and some metabolic enzymes. In duck lens, two crystallins had very high metabolic enzyme activities： (1) ε-Crystallin was shown to possess L-lactate dehydrogenase activity. (2) δ -Crystallin was shown to possess argininosuccinate lyase activity. This thesis was divided into three sections for study (A) The preparation and purification of duck crystallins Duck eyeballs were freshly collected, decapsulated, homogenized,and centrifuged. The supematant was crude duck crystallin. ε-Crystallin arid δ- crystallin were partially purified by gel filtration chromatography. ε-Crystallin was well purified by ion exchange chromatography. But δ- crystallin was partial purified by ion exchange chromatography. Then, the purity of purifiedε-crystallins was characterized by gel electrophoresis and high performance liquid chromatography. (B) The study of enzymatic activity of duck ε-crystallin The thermostability of ε-crystallin enzymatic activity was determined at two different temperatures. Both crude and purified ε-crystallin enzymatic activity retained above 60% at 25℃ in 21 days. Purified ε-crystallin still retained enzymatic activity at 62℃ in 8 hours. These results showed that ε-crystallin enzymatic activity was very stable. Then, various 2-keto acids were served as the substrates of ε-crystallin. The substrate binding site of ε-crystallin was probed by the reaction properties of various 2-keto acids. The result indicated that the substrate binding site of Ecrystallin was significantly different from that of other source L-lactate dehydrogenases. Because ε-crystallin possessed very stable enzymatic activity, it could be served as a biocatalyst for the syntheses of chiral 2-hydroxybutyric and 2-hydroxypentanoic acids from 2-ketobutyric and 2-ketopentanoic acids, respectively. 2-Hydroxytbutyric and 2-hydroxy-pentanoic acids were the precursors of insect pheromones and fungicides, respectively. The result showed that ε-crystallin was very suitable for serving as a biocatalyst for synthesis of valuable chiral compounds. (C) The study of enzymatic activity of duck δ-crystallin The substrate binding site of δ -crystallin was probed by the reaction properties of various substrate analogues. The result indicated that the substrate binding site of δ -crystallin was highly restrained for its substrates and just could accept very few substrates. Because δ -crystallin possessed stable enzymatic activity, it could be served as a biocatalyst for the synthesis of L-argininosuccinate from L-arginine and fumarate. L-Argininosuccinate could be served as the substrate of argininosuccinate lyase to diagnose whether the argininosuccinic acduric was occurred. The result showed that δ -crystallin could synthesize argininosuccinate efficiently and was reuable. For δ -crystallin, the main defect was that it just could accept very few substrates and couldn’t synthesize many kinds of products.