Purification and characterization of an alpha-2-macroglobulin protease inhibitor from plasma of the grouper Epinephelus coioides

An alpha-2-macroglobulin (α-2-M) of the grouper Epinephelus coioides was purified by Fast Protein Liquid Chromatography with various columns including Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Sepharose 4B and Phenyl Sepharose High Performance. The purified protein electrophoresed as a single protein band in both native PAGE (380 kDa) and non-reduced SDS-PAGE (180 kDa) while electrophoresed as two protein bands (97 and 80 kDa) in reduced SDS-PAGE. In addition, the purified protein was a glycoprotein as it could be visualized by using a glycoprotein staining kit in both non-reduced and reduced SDS-PAGE. As measured by trypsin-N-benzoyl-dl-arginine-p-nitroanilide assay, the protease inhibitory activities of grouper plasma and the purified protein decreased to 27 and 17%, respectively, at 60 °C, and increased at pH 7.0 to 11.0, and decreased when concentration of methylamine increased. From the above characterizations, the purified protein was confirmed to be an α-2-M. This study is for the first time to describe the presence, purification and characterization of an α-2-M from the grouper.