Purification and immunological determination of α₂-macroglobulin in serum from injured rats

Rat α₂-macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose, and gel filtration chromatography. The final protein product obtained by this procedure proved to be α₂-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The α₂-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat α₂-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170 000-dalton α₂-macroglobulin subunit. The concentration of α₂-macroglobulin in the sera of rats was measured by electroimmuno assay using a monospecific antiserum against α₂-macroglobulin. Purified α₂-macroglobulin was used as a standard. Sera from normal male rats contained 32 ± 4 μg of α₂-macroglobulin per ml. To determine the time course of response of α₂-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of α₂-macroglobulin increased rapidly, reaching a maximum value of 110-140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.