Human haptoglobins: estimation and purification

Abstract

A procedure for the rapid, accurate estimation of haptoglobin in serum and purified preparations is described, based on the peroxidase activity of hemoglobin in its complexes with haptoglobin. Hydrogen peroxide, guaiacol and methemoglobin reagents are used, under well-defined conditions which reduce the peroxidase activity of free hemoglobin essentially to zero. The reaction is followed spectrophotometrically. The method is calibrated so that the hapto- globin content of a solution can be expressed in terms of its hemoglobin- binding capacity. Haptoglobin has been prepared in high purity from 1 to 150 ml of serum in approximately 50% yield, by bulk-adsorption from dialyzed serum at pH 4.2 with Dowex 2 X-10 anionic-exchange resin in the chloride form, followed by washing in a column and elution with 0.05 M NaCl solution. Purified haptoglobins from sera of the 3 common haptoglobin types and from serum of the modified type, Hp 2-1 (Mod.), have been examined by starch-gel electrophoresis. Evidence is presented that the haptoglobins obtained are undamaged by the purification, and that the many individual haptoglobin components demon-strated in the several genetic types are not artifacts.