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PT07 Chemical and Biological Analysis of Endocrine Disrupting Compounds
(PT121) Isolation and Characterization of California Halibut (Paralichthys Californicus) Vitellogenin for Use in a Sediment Bioassay.
Palumbo, A1, Linares-Casenave, J1, Tjeerdema, R1, 1 University of California, Davis, CA, USA
ABSTRACT- Population declines of aquatic wildlife have been correlated to environmental contaminants that are capable of modifying hormone actions. To evaluate estrogen-mimicking contaminants in sediments along the California coast, a bioassay is being developed using the benthic flatfish, California halibut (Paralichthys Californicus). It consists of quantification of an estrogen responsive egg yolk protein, vitellogenin (Vg) in halibut plasma. In order to develop the assay, Vg was isolated and characterized from California halibut. Male fish were injected with 1mg/kg 17−estradiol and plasma was collected after two weeks. Vg was purified from plasma by size exclusion and ion exchange chromatography. SDS PAGE and western blot showed one major putative Vg band (180 kD) in the plasma sample and the same band plus 2 others, likely subunits of Vg (120 and 80 kD), in the purified solution. Native PAGE and western blot revealed the same profile in both plasma and purified Vg samples, suggesting that the purification was successful, and that the degradation of the purified Vg in the SDS PAGE was due to the harsh conditions of that technique. Molecular mass determination by MALDI TOF resulted in a large peak at 188 kD. Mass Spectra of peptide sequences from liquid chromatography tandem mass spectrometry confirmed identity of the protein by matching with 5 Vg sequences from plaice (Pleuronectes platessa) and 3 from European flounder (Platichthys flesus). The Vg is being used to develop a quantitative enzyme-linked immunosorbent assay (ELISA) for California halibut Vg. This bioassay will be used to detect estrogen mimics in sediments along California's coastline.
Key words: isolation, vitellogenin, environmental estrogens, Paralichthys Californicus
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