title: Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography.
abstract: Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.
authors.affiliation: Department of Biological Science and Technology, Research Institute of Biochemical Engineering, National Chiao Tung University, Hsinchu, Taiwan, ROC.
authors.forename: Chi Feng
authors.initials: CF
authors.lastname: Tseng
authors.forename: Hsing Yi
authors.initials: HY
authors.lastname: Huang
authors.forename: Yuan Ting
authors.initials: YT
authors.lastname: Yang
authors.forename: Simon J T
authors.initials: SJ
authors.lastname: Mao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000917:Antibody Formation; D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D006041:Goats; D006242:Haptoglobins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits
chemical_list: D000911:Antibodies, Monoclonal; D006242:Haptoglobins; D021122:Protein Subunits