單一型態Haptoglobin純化與分析

Abstract

人類Haptoglobin (Hp)可區分為三種表現形：Hp 1-1, 2-1, 2-2。然而，Hp 2-1和2-2 (不包括Hp 1-1) 本質上具有較複雜的多形性與異質性，使其具有不同的分子大小。人類Hp 1-1、鹿Hp以及豬Hp在型態上個別為同質性二聚體 (□□)2、四聚體 (□□)4和二聚體 (□□)2。本篇報告中，我們建立了一個簡單純化具有同質性但不同物種Hp的純化方式。一開始先將血漿以50%飽和硫酸銨沉澱，取其上層液通入高效液相層析儀搭配凝膠過濾管柱(gel-filtration column)並以0.3 ml/min 磷酸生理食鹽水沖提。發現豬與鹿Hp的純度可達90%以上，但是在純化的人類Hp 1-1有脂蛋白原A-1(apolipoprotein A-1；apoA-1)及血清白蛋白(albumin)存在。因此針對去除脂蛋白原A-1與血清白蛋白提出了「策略性」的解決方法，以去除脂蛋白的血漿當純化起始物並搭配銅離子結合固定化金屬離子親和層析法{Cu (II)-immobilized metal ion affinity chromatography；Cu (II)-IMAC}搭配純化人類Hp 1-1，其純度可達95%以上。以不連續膠體電泳(SDS-PAGE)與西方點墨法(Western blot)描繪純化後的蛋白質分子量形式。純化後的Hp保有原本可和血紅素(Hemoglobin)結合的能力，證實了純化過程中並沒有改變其蛋白質結構。最後在本篇報告中亦討論個別純化步驟中的純度(purity)與產率(yield)。

Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. However, human Hp 2-1 and 2-2 (except 1-1) are polymeric and heterogeneous in nature with various molecular sizes. Human Hp 1-1, cervine Hp, and porcine Hp is homogeneous dimer (□□)2, tetramer (□□)4, and dimer (□□)2, correspondingly. This report provides a simple protocol that can be used to isolate the homogeneous Hp from different species. Plasma was first fractionated using a 50% saturated ammonium sulfate. The supernatant was then chromatographed on a HPLC gel-filtration column equilibrated with PBS (pH 7.4) at a flow rate of 0.3 ml/min. The homogeneity of isolated porcine and cervine Hp was greater than 90%, but the contamination of apolipoprotein A-1 (apoA-1) and albumin existed in that of human Hp 1-1. We proposed a “strategic solution” for the elimination of apoA-1 and albumin by using lipoprotein depleted plasma as a starting material and Cu (II)-immobilized metal ion affinity chromatography (IMAC), respectively. Finally, a purity with 95% homogeneity of human Hp 1-1 was achieved. The molecular form of isolated human, deer, and pig Hp was further characterized by SDS-PAGE and Western blot. Each isolated Hp species still preserved the hemoglobin binding ability suggesting that the purification procedure did not alter the native structure of Hp. The recovery of Hp in each isolation step is also described and discussed.