The α macroglobulins of rat serum

Abstract

A three-stage method for isolation of α1 macroglobulin and α2 macroglobulin from the serum of normal and injured rats is described. The methods successively used, namely gel filtration, ultracentrifugation and chromatography on DEAE-cellulose, were chosen to minimize loss of tryptic esterase-protecting activity. The two proteins differed slightly with respect to the following properties: mol.wt., α1 macroglobulin 7.46 × 10(5), α2 macroglobulin 7.16 × 10(5); isoelectric focusing, α1, macroglobulin pI 4.4, α2 macroglobulin pI4.5. Amino acid analyses were identical, except with respect to tyrosine: α1 macroglobulin 3.96 ± 0.24, α2 macroglobulin 3.16 ± 0.32 mol/100 mol of total amino acids. When isolated from the serum of uninjured rats, α1 macroglobulin retained the capacity to bind 1.05 mol of trypsin/mol. However, if isolated from serum 2 days after injury only 0.78 mol of trypsin/mol of α1 macroglobulin was bound. α2 macroglobulin isolated from this latter serum bound on average 0.97 mol of trypsin/mol. When reduced with N-acetylcysteine, both molecules formed subunits of size corresponding to that expected for quarter molecules. When α2 macroglobulin was reduced with dithiothreitol, quarter molecules were again produced. α1 macroglobulin, however, when thus treated gave a more complex mixture, containing a component having a mol.wt. of less than 6 × 10(4). Antisera raised against the two proteins permitted estimation of the concentration of each protein in the plasmas or sera of normal and injured rats. Plasma from normal male rats contained 3.76 ± 0.56 mg of α1 macroglobulin/ml (n = 33) and 0.016 ± 0.001 mg of α2 macroglobulin/ml (n=33). After injury by injection of turpentine and cortisone, the concentrations in plasma were at 3 days 5.19 ± 0.81 mg of α1 macroglobulin/ml (n = 12) and at 2 days 1.38 ± 0.35 mg of α2 macroglobulin/ml (n = 12). Antisera to the two proteins did not cross-react with one another. The quarter molecules formed by reduction of both proteins showed increased antigenicity.