苦茶子中溶血因子的分離與活性分析
中文摘要
從苦茶種子中用 5% 醋酸萃取出具有溶血活性的物質。這溶血因子經過 DEAE-Sephacel (D-1) 及 SephandexG75(S-2) 管住及聚丙烯胺柱狀交體 (P-1) 分離後，得到一溶血活性比 較酸萃取物增加 28 倍的溶血因子，而產率微酸萃取物之 27.58% 。經由平板式不連續 硫酸十二酯鈉 - 聚丙烯胺膠體電泳 (10%) 分析 P-1 之分子量，用硝酸銀染色發現一條較 深色的色帶其分子量約 58.8kD ，其他染色較微弱的色帶則為 61.8kD 及 43.5kD 。
溶血因子的活性不受溫度的影響，置於 100 度的水浴鍋中煮沸一小時，發現其溶血活性
沒有改變；暴露在低於 10 度的低溫下，則會降低其溶血活性。加入蛋白變性試劑如 :8 M urea 、 B-mercaptoethanol 、 proteinase K 、 trypsin 、 pepsin 、 NaOH 及 HCI 等破壞 分子結構，結果顯示溶血活性不被抑制，仍具有對照組 95% 的溶血因子。薄層色層分 析出 S-2 溶血因子具有 aminodeoxyhexose 的成分。
溶血因子可因加入 Fucose 、 Ribose 、 Fructose 、 Galactosamine 及 ConA 而有抑制溶血 的現象，這表示溶血作用是經由辨識紅血球細胞膜的醣脂質或醣蛋白結構。過去的結果 顯示此溶血因子作用並不是皂素所引起，也不是因磷脂酶的活性作用。溶血因子不會使 鼠腦 MDA 的濃度增加，意味著溶血作用不經由脂質過氧化反應。
整體來說，溶血因子可以被部分純化並且顯示對高溫及強變性試劑的耐受性。從溶血機
轉來討論，目前並不清楚。不過這反應可能是辨識紅血球細胞膜表面的醣脂質或醣蛋白 上的醣分子，而且不伴隨著磷脂酶降解或行程自由基的關係。
The Isolation and Characterization of the Hemolysis factor from
英文摘要
The hemolysis factor was isolated from the seed extract of Camellia japonica L. and purified b y DEAE-Sephacel column(D-1),sephadex G75
Column(S-2) and polyacrylamide disc gel(P-1). The purity of S-2 fraction exhibited a 28-fold increase according to the hemolysis activity and the yield is 27.58% of crude extract. The mol ecular weight of P-1 fraction was determined by SDS-PAGE and found that there are one maj or band (58.8kD)and two minor bands(61.68and43.54kD)by using the silver staining.
The hemolysis factor was also characterized as thermal-resistant ； hoeever, the hemolysis act ivity was inhibited by lower temperature. To treat with structure-disrupting agents, it revealed that the hemolysis factor is highly resistant to urea, B-mercaptoethanol, proteinase K, trypsin a nd pepsin. Analysis of silica thin layer chromatography indicated that S-2 contains aminodeox yhexose.
The hemolysis ability was inhibited by addition of fucose, ribose, fructose, galactosamine and Con A, indicating that the hemolysis was via the recognition of the surface of sugar moieties.
Previous results indicated hemolysis factor is not a saponin and did not exhibit phospholipase activity. Incubation of hemolysis factor with brain lipid did not increase the production of MD A(TBA)2, suggesting that the hemolysis was not mediated by radical formation.
Overall, hemolysis factor was partially purified and exhibit the properties of susceptibility of h igh temperature and strong denaturants. The mechanism of hemolysis was not really clear ； h owever, the reaction was mediated by the recognition the sugar moieties of surface which is n ot accompanied with the phospholipase degradation and free redical formation.
