Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction, isolation and partial characterization

Abstract

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17β in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17β treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl2, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.