Isolation and characterization of alpha 2-macroglobulin from mastitis milk

Whey samples were screened for the presence of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). From an enzymic test, alpha 2M levels in normal whey varied in the range 0.49-0.84% of the serum level, whereas in mastitis whey the activity was markedly increased, reaching values between 0.91 and 138.5% (median 7.2%) of standard serum level. In mastitis milk samples but not in normal milk alpha 2M was also detected by double immunodiffusion and Western blotting. The proteinase inhibitor was purified from a mastitis milk sample with high alpha 2M activity (138.5% of serum level). In SDS-PAGE, native-PAGE and in double immunodiffusion analysis the inhibitor appeared indistinguishable from plasma-derived alpha 2M. The alpha 2M preparation from mastitis whey migrated essentially as native alpha 2M, representing the 'slow' form of the molecule. Treatment with trypsin transformed the alpha 2M preparation to the electrophoretic 'fast' form, but treatment with methylamine had only a minor effect. The receptor recognition sites were not exposed on the isolated alpha 2M molecule but could be readily exposed by treatment of the proteinase inhibitor with trypsin, which further proved that the isolated alpha 2M was in the entire native, functionally active state.