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  • Title: Use of immobilized metal ion affinity chromatography and dye-ligand chromatography for the separation and purification of rainbow trout pituitary gonadotropins, GTH I and GTH II.
    Author: Govoroun MS, Huet JC, Pernollet JC, Breton B.
    Journal: J Chromatogr B Biomed Sci Appl; 1997 Sep 26; 698(1-2):35-46. PubMed ID: 9367191.
    Abstract:
    A new procedure is described for the purification of gonadotropic hormones (GTHs) from the pituitary glands of vitellogenic rainbow trout. The procedure utilizes immobilized metal ion affinity chromatography (IMAC) on a column containing immobilized iminodiacetic acid (Toyopearl AF Chelate) charged with Cu2+ ions as a critical step for the efficient separation of GTH I and GTH II. Further purification of both GTH fractions on Cibacron Blue F3GA immobilized on Toyopearl was followed by HPLC size-exclusion for GTH II. The resulting electrophoretically homogeneous preparations possessed a characteristic range of biological activity in the stimulation of steroidogenesis in vitro. N-Terminal sequences of the GTH I and GTH II subunits purified using reversed-phase HPLC revealed a high level of homology with those of GTH subunits found in three other salmonid fish species. In contrast to salmon GTH II, two different alpha-subunits were observed in trout GTH II. The cross-reactivity between GTH I and GTH II was studied in radioimmunoassay using antibodies against Chinook salmon GTH II beta-subunit and rainbow trout GTH I dimer.
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