Purification and Fractionation of Rat Haptoglobin: Characterization and Comparison with Human Haptoglobin

Abstract

A three step procedure for the isolation of haptoglobin from the serum of turpentine inflamed rats is described. Subsequent to purification, cleavage of the purified rat as well as human haptoglobin into its substituent alpha and beta subunits is performed. The three steps successfully used in haptoglobin purification were: gel filtration on G-200 sephadex, stepwise gradient ion exchange chromatography, and repeated gel filtrations on G-100 sephadex. The use of chromatographic hydroxylapatite proved unsuccessful for serum haptoglobin purification. Interchain disulfide bond cleavage followed by two-iodoacetamide alkylation of the separated haptoglobin alpha and beta subunits permitted their dispersal and separation through a column of G-75 sephadex. Identification and characterization of the purified protein was performed using SDS acrylamide gel electrophoresis, thin-layer gel electrofocusing, enzymatic determination, statistical manipulation of the amino acid analysis data, and immunoelectrophoresis. Inherent substituent composition for the purified protein and their respective chains were determined by amino acid analysis, carbohydrate analysis, and enzymatic digests. Antibodies to the purified rat haptoglobin were developed in rabbits. Human and rat haptoglobins were seen to demonstrate distinctly different molecular characteristics regarding their microheterogeneity upon thin layer polyacrylamide gels and mobilities within SDS acrylamide gels. The substituent composition for these two mammalian haptoglobins is seen to differ via amino acid analysis and sugar content. More specifically, the purified alpha chains of rat and human haptoglobin share no common tryptic and chymotryptic cleavage sites. Similarities do exist for these two mammalian haptoglobins regarding their principle physiologic function, nature of basic subunit composition, and demonstration of polymerism.