Purification and characterization of vitellin from the freshwater giant prawn, Macrobrachium rosenbergii

Vitellin of Macrobrachium rosenbergii was purified by the combined use of ultrafiltration, ion exchange high-performance liquid chromatography (HPLC), and PAGE. The vitellin fraction recovered from HPLC contained 4 proteins, B1-B4, each of which was composed of 2 major polypeptides (A and B), as well as other minor components (E and F polypeptides and others larger than 205 kDa in molecular weight) on SDS-PAGE. Polymorphism of vitellin is clearly demonstrated, and the E polypeptide was found to be the possible precursor of A and B polypeptides as revealed by peptide mapping followed by Western blotting. The molecular weights of A, B, and E polypeptides were estimated by SDS-PAGE to be 83.1, 88.8, and 147.8 kDa, respectively, and those of A and B polypeptides were further characterized by MALDI-TOF mass spectrometry to be 95.9 and 106.9 kDa. The vitellin components were associated with each other through non-disulfide bonds as revealed by non-reducing SDS-PAGE and MALDI-TOF mass spectrometry. Hemolymph vitellogenin (Vg) was composed of 3 polypeptides, i.e., vc1, vc2, and vc3. Purified vitellin is immunologically and electrophoretically identical to Vg. A and B polypeptides are immunochemically correlated with vc1 and vc3, and vc2 and vc3, respectively; vc3 could therefore be the possible precursor of vc1 and vc2. Partial amino acid sequences of A and B polypeptides are also presented, and the transformational processing of Vg into vitellin is further discussed.