Identification, Purification, and Immunoassay of Stone Flounder (Kareius bicolouratus) Vitellogenin

Vitellogenin (Vtg), the precursor of egg yolk proteins, serves as nutrients for the developing embryos. Vtg production in male fish has become an excellent biomarker for environmental estrogens. Thus, Vtg synthesis was induced by injection of 17 beta-estradiol (E-2) in stone flounder (Kareius bicolouratus). Plasma from uninduced and E-2-treated fish was subjected to native polyacrylamide gel electrophoresis (PAGE) and the gels were stained with Coomassie brilliant blue R250 for proteins, methyl green for phosphates, Sudan black B for lipids, and periodic acid-Schiff's reagent for carbohydrates. These analyses verified the presence of two phospholipoglycoproteins in the plasma from E-2-treated stone flounder, which were tentatively identified as Vtg. Vtg was then isolated by two purification procedures: gel filtration followed by anion exchange chromatography, or selective precipitation combined with anion exchange chromatography. The latter procedure was more effective at isolating Vtg with a high degree of purity. In native PAGE, the apparent molecular masses of Vtgs were determined to be 520 and 550 kDa, respectively. Two major bands with approximate molecular masses of 165 and 106 kDa together with two additional faint bands of lower molecular weights were observed after sodium dodecyl sulfate-PAGE under reducing conditions. These bands exhibited differential affinity for polyclonal antisera against red drum (Sciaenops ocellatus) Vtg in Western blot. The present study established the purification procedure and characterized the electrophoretic and immunological properties of stone flounder Vtg.