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研究生(外文):Liao Woan-Ru
論文名稱(外文):studies on the agglutinins of marine algae from Taiwan
指導教授:黃 穰林榮耀林榮耀引用關係
指導教授(外文):Huang RangLin Jung-Yaw
外文關鍵詞:Agglutininsmarin macroalgaemicroalgaehemagglutinationisolated and purificationGalaxaura marginata lectinenzyme tretreatmentmedical application
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摘 要
凝集素廣泛地存在於自然界不同生物體內,具有凝集紅血球及其他正常或變形細胞的能力,常被應用於細胞表面癌症辨識物及其他生物醫學的研究。本論文研究是從台灣沿岸採集之海洋大型海藻與分離培養的海洋微細藻類共三十種進行凝集活性檢測,並對具高活者藻類進行分離純化。以磷酸鹽緩衝液及20 %乙醇分別萃取出海藻並以粗萃取液對人類A、B、O、AB血型、凝集缺陷患者以及大白鼠、綿羊及猴子三種動物的紅血球進行凝集活性檢測。結果發現在大型海藻中紅藻類之麒麟菜、綠藻類之裂片石蓴及褐藻類的重緣馬尾藻,具有較高之凝集力價達212 titer,而紅藻的龍鬚菜凝集力價較低,但具抗B型紅血球活性。微細藻類則普遍有較高凝集力價。以鏈蛋白酵素( pronase )或胰蛋白酵素( trypsin )處理人類及動物紅血球表面。大多提高粗萃取液的凝集活性8~64倍以上。在幾種測試的微細藻類其萃取液不需2價陽離子才具活性。海藻萃取液在4℃ 和 -20 ℃中經貯存6~12個月後,活性明顯降低。以鋸齒麒麟菜為例,酵素作用適合時間為2~6小時,在45 ℃下維持原有活性,但為不耐高溫種類。以DEAE Column分離純化出的鋸齒麒麟菜凝集素(ESA)蛋白質含量為每1g乾重藻體有10.2 mg。以SDS- PAGE凝膠電泳法得到該藻凝集素之分子量為29,000。醣抑制試驗結果顯示該凝集素對許多單醣及寡糖均有親和性。篩選過程中亦分離純化扁乳節藻的凝集素,具毒蛋白之溶血作用特性,分子量為26,000 Kda,該凝集素醣抑制試驗結果顯示該凝集素,對許多單醣及寡糖均有親和性。本論文亦利用西方點墨法初步篩選並發現藻類不具有抗癌功能的植物凝集素中。基於高凝集活性,海洋大型及微細藻類可做為研究親醣蛋白的生化及醫藥用途的材料。此外凝集素可能在藻類生物及生態上扮演重要角色。
Agglutinins are widely distributed in nature. They are capable of agglutinating erythrocytes and other normal or transformal cells and often used in the studies for biomedical purposes. In this study, thirty species of marine macroalgae, collected from the northeastern coast of Taiwan, and marine unicellular algae were assayed for hemagglutination, followed by isolation and purification of agglutinins in the selected algae. Algal extracts in both phosphate buffer saline (PBS) and aqueous ethanol were assayed for agglutination against erythrocytes of both human normal blood groups, i.e. A,B,O,AB, and blood with coagulation disorder as well as against rate, sheep and monkey erythrocytes. The results indicate that all of the marine algae assayed can agglutinate human and animal erythrocytes. Of the macroalgae, Eucheuma serra (red alga), Ulva fasciata (green alga) and Sargassum duplicatum (brown alga) are most prominent for such agglutination, reaching 212 titers. Extract of Gracilaria lemaneiformis (red alga) is less active in hemagglutination, but it has strong afinity for B erythrocytes. Marine microalgae also contain agglutinins, with activity generally higher activity than that for macroalage.
Tretreatment of erythrocytes with pronase or trypsin for 2-6 hr improves markedly the detection of algal activity in general. Moreover, algal extracts remain active in agglutination at 45 ℃. The assayed microalgae have no requirement for divalent cations in hemagglutination. Agglutinating activity of crude algal extracts decreased markedly during the storage at 4 ℃ and —20 ℃ for 6-12 months.
The agglutinin of Eucheuma serra (ESA) was isolated by DEAE Column, containing 10.2 mg protein/g dry wt of algal powder, with a band of 29,000 on SDS-PAGE. ESA has the specific binding affinity for mono- and oligosaccharides. Moreover, the isolated protein from the red alga Galaxaura marginata demonstrates hemolysis activity, with molecular weight of 26,000 Kda. The protein also binds mono- and oligosaccharides. Further assay was employed for the detection of plant glycoprotein in algal extracts using antibodies of wheatgerm agglutinin (WGA) and concanavalin A (Con A) in ELISIA plate and western blotting.
In light of the generally strong activities, both marine macroalgae and microalgae are a potent source of lectins for study in biochemistry and medical applications. Some of the algal agglutinins probably have played important roles in algal biology and ecology.
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