番木瓜種子凝集素之純化與生化特性探討

Abstract

本研究利用先前發表之酵素聯結親和吸附分析法（Food Chem. 2009, 113: 1218-1225），以聚丙烯醯胺聯結N-乙醯半乳糖胺（GalNAc）做為篩選凝集素之介質，發現木瓜子中含有與GalNAc專一結合之凝集素。檢驗不同成熟度之木瓜子與凝集素含量的關係，發現青木瓜中未成熟之白子具有較高的凝集素比活性。本研究依下列方式分離木瓜子凝集素：首先將樣品冷凍乾燥後磨碎過篩（40 mesh），以20倍（v/w）PBS buffer（pH 7.4）於4oC萃取隔夜，離心後取上清液加硫酸銨至70%飽和度，離心取沉澱回溶透析後，再經超過濾進行分子量區分。將分子量大於50 kDa的區分，以HiTrap CM FF進行離子交換層析及Superdex 200 GL膠體過濾區分後，分離得純化之凝集素（Carica papaya lectin, CPL）。SDS-PAGE結果顯示，於38及40 kDa分別有一明顯的色帶；Shodex KW-804膠體過濾層析顯示，原態CPL的分子量為804 ± 30 kDa，推測CPL應由α、β兩個次單元組成的多聚體。溫度及酸鹼安定性方面，CPL在70oC會失活，且於pH 6.0~8.0之間皆保有最高的活性。以EDTA和Ca2+、Mg2+、Mn2+、Zn2+處理CPL，發現其不需要二價金屬離子來維持活性。醣類抑制試驗結果指出，CPL對GalNAc有顯著特異性，其次是lactose。對人類不同血型紅血球的凝集能力結果顯示，CPL對A型血球具有較高的凝集活性。另外，以自行衍生的GalNAc-Sepharose 6B親和性管柱來純化CPL，可提高其純度達粗萃液的6000倍。

Based on our previous study, a novel N-acetylgalactosamine binding protein in papaya seed (Carica papaya lecin, CPL) was discovered by GalNAc-polyacrylamide based enzyme-linked adsorbent assay (Food Chem. 2009, 113: 1218-1225). Papaya seeds with different maturity were collected, and the extracts of seeds in green papaya fruits shows relatively high activity. Further purification of CPL was conducted as following: freeze-dried papaya seeds were ground into powder, extracted with 20 folds (w/v) of 50 mM phosphate buffered saline (pH 7.4) at 4°C overnight, centrifuged and collected the supernatant, added ammonium sulfate to 70% saturation. Precipitations were resuspended and dialyzed against PBS, then fractionated by a 50 kDa MWCO ultrafiltration. The retentate was further purified by HiTrap CM FF ion exchange chromatography and Superdex 200 GL gel filtration chromatography. SDS-PAGE and HPLC gel filtration indicated that CPL is a polymer with a molecular mass of 804 ± 30 kDa and composed of two different subunits of 38 and 40 kDa associated by non-covalent bonds. It was heat stable until up to 70oC for 30 min and showed optimum sugar-binding activity from pH 6.0 to 8.0. Also, CPL did not require Ca2+, Mg2+, Mn2+, Zn2+for its activity. Of various sugars tested, the lectin was best inhibited by GalNAc. CPL agglutinated all trypsinized human RBC types, with a slight preference for the A blood group which immunodeterminant is GalNAc. Based on the specificity, we performed an alternative ripid, simple purification method via GalNAc-Sepharose 6B affinity chromatography column, and gave the lectin with a 6000-fold purification as compared to the crude extract.