Isolation and characterization of plasminogen and plasmin from bovine milk

Abstract

Abstract A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino acid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation contained the proteolytically modified midi-plasmin, consisting of kringle 4,5 and the light chain with an apparent molecular weight of 50 k Da in unreduced SDS-PAGE, and resulting in two bands of 30 and 26 k Da after reduction. Some 15% of the amino acid sequence of plasminogen/midi-plasmin isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated from bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 267–278), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593–603). Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen associated with the milk fat globule membrane was very low and reflected the presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85, 80 and 50 k Da in unreduced gels. These hands may represent two plasminogen forms (85 and 80 k Da) and midi-plasmin/midi-plasminogen (50 k Da). The total concentration of plasminogen in bovine milk was estimated to 1.5 μg/mL.