Vol. 72 ESTIMATION AND PURIFICATION OF HAPTOGLOBINS

1. A procedure for the rapid, accurate estimation of haptoglobin in serum and purified preparations is described, based on the peroxidase activity of haemoglobin in its complexes with haptoglobin. 2. Hydrogen peroxide, guaiacol and methaemoglobin reagents are used, under well-defined conditions which reduce the peroxidase activity of free haemoglobin essentially to zero. The reaction is followed spectrophotometrically. 3. The method is calibrated so that the haptoglobin content of a solution can be expressed in terms of its haemoglobin-binding capacity. 4. Haptoglobin has been prepared in high purity from 1 to 150 ml. of serum in approximately 50% yield, by bulk-adsorption from dialysed 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 0-05M-sodium chloride solution. 5. Purified haptoglobins from sera of the three common haptoglobin types and from serum of the modified type, Hp 2-1 (Mod.), have been examined by starch-gel electrophoresis. 6. Evidence is presented that the haptoglobins obtained are undamaged by the purification, and that the many individual haptoglobin components demonstrated in the several genetic types are not artifacts.