Analysis and isolation of single type haptoglobin

• Main Authors: Kuan Yu Lin, 林冠佑
• Other Authors: Simon J. T. Mao
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/68042303013350651514

碩士 === 國立交通大學 === 生化工程研究所 === 95 === 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.