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Purification and immunological determination of alpha 2-macroglobulin in serum from injured rats
Rat alpha 2-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 alpha 2-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The alpha 2-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat alpha 2-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170,000-dalton alpha 2-macroglobulin subunit. The concentration of alpha 2-macroglobulin in the sera of rats was measured by electroimmuno assay a monospecific antiserum against alpha 2-macroglobulin. Purified alpha 2-macroglobulin was used as a standard. Sera from normal male rats contained 32 +/- 4 micrograms of alpha 2-macroglobulin per ml. To determine the time course of response of alpha 2-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of alpha 2-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.
Rat alpha 2-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 alpha 2-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The alpha 2-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat alpha 2-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170,000-dalton alpha 2-macroglobulin subunit. The concentration of alpha 2-macroglobulin in the sera of rats was measured by electroimmuno assay a monospecific antiserum against alpha 2-macroglobulin. Purified alpha 2-macroglobulin was used as a standard. Sera from normal male rats contained 32 +/- 4 micrograms of alpha 2-macroglobulin per ml. To determine the time course of response of alpha 2-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of alpha 2-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.