雞蛋卵清蛋白與大頭鰱性腺刺激素次單元之寡醣結構研究

Abstract

中文摘要 本實驗的目的是分離雞蛋卵清蛋白（ chicken egg ovalbumin ）和大頭鰱(Hypophthalmichthys nobilis; Bighead carp ）腦下腺性線刺激素（ Gonadotropin , GTH ) α、β次單元的寡醣並研究其結構。 卵清蛋白經 Trypsin 水解以 Concanavalin A ( ConA ) - Sepharose affinity column 分離 glycopeptides ，部分 glycopeptides 利用 Reverse－phase HPLC ( Lichrosorb RP C8 column ) ，分離最短 glycopeptide ，胺基酸定序決定糖化位置（ glycosylation site ）為 Asn 293。 其餘 glycopeptides 以 N – glycosidase F ( PNGase F）切除寡醣，得到的寡醣混合物經過螢光標示（使用螢光試劑2－ aminopyridine ）後，以 gel-fil - tration ( HW40 column ）及 HPLC ( PALPAK type R column ）分離各個 Pyridylamino ( PA-）寡糖。 大頭鰱腦下腺粉末以 Hartree 溶液萃取得粗醣蛋白，利用 DEAE－cellulose column ，不同濃度碳酸氫銨循序滌流分離得到四部份（ DE 1－4 ) , reverse－ phase HPLC 解析 DE 1－4 ，以氨基酸組成分析及定序鑑定，結果於 DE1 發現性腺刺激素遊離α次單元； DE2發現性腺刺激素（α、β dimer ) ; DE3 、 DE4 發現性腺刺激素遊離β次單元，收集遊離α、β次單元，經 PNGase 切除寡糖得到寡糖混合物，經過螢光標示後，以 gel-filtration 和 HPLC 分離各個 pyridylamino ( PA-）寡醣。

Abstract The present study was aimed to set up the procedures for the determination of oligosaccharides present in the glycoprotein. So, chicken egg ovalbumin and bighead carp gonadotropin were selected for the purpose. Ovalbumin was digested by trypsin, and the glycopeptides were purified by Con A-sepharose chromatography from the trypsin digestion. Using reverse-phase HPLC, a glycopeptide was found to fit the expected amino acid composition and its amino acid sequence shows that the glycosylation site is at the position Asn293 . Preparation of oligosaccharides from glycopeptides by N-glycosidase digestion, and derivatization of the oligosaccharides with a fluorescent reagent, 2-aminopyridine. The pyridylamino (PA-) oligosaccharides were purified by gel-filtration (HW40 column) and HPLC (PALPAK type R). Bighead carp pituitary glands were extracted with Hartree solution. The crude glycoprotein was then chromatographed by DEAE-cellulose column, Four fractions were obtained (DE1-4), DE1—4 were purified further by reverse- phase HPLC and characterizated by amino acid composition and sequence analysis. Gonadotropin (GTH) free α subunit; α、β dimer and free β subunit were isolated respectively from Del、 DE2 and DE3/DE4. Preparation of oligosaccharides from glycoprotein (free α subunit and free β subunit) by N-glycosidase digestion, and derivatization of the oligosaccharides with a fluorescent reagent,2-aminopyridine. The pyridylamino (PA-) oligosaccharides was purified by gel-filtration and HPLC.