Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography.
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.
Tseng
Chi Feng
CF
Department of Biological Science and Technology, Research Institute of Biochemical Engineering, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Huang
Hsing Yi
HY
Yang
Yuan Ting
YT
Mao
Simon J T
SJ
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
D000911:Antibodies, Monoclonal; D006242:Haptoglobins; D021122:Protein Subunits