Human pituitary thyrotropin. Isolation of alpha and beta subunits by hydrophobic interaction chromatography.

ABSTRACT

Various conditions for the dissociation of highly purified human pituitary thyrotropin into subunits have been investigated. Dissociation on a preparative scale was accomplished by treatment with 1 M propionic acid at 32 degrees C for 16 h. The isolation of one alpha and two beta subunits was achieved by hydrophobic interaction chromatography on pentyl-Sepharose-4B. Radioimmunological technique was utilized to classify the subunits in accordance with current nomenclature and also to express their activities. The activities of the subunits overlapped insignificantly (less than or equal to 0.3%) and their content of intact thyrotropin was negligible (less than 0.05%). The characterization of the subunits included determination of their amino acid compositions. Analytical polyacrylamide gel electrophoresis of the subunits at acid and alkaline pH values revealed heterogeneity. By free-zone electrophoresis at alkaline pH it was possible to isolate four discrete iso-forms of both the alpha and the beta subunit. All these eight individual subunits had activities consistent with those of their immediate precursor fractions. Isolation of electrophoretically homogeneous thyrotropin subunits has not been reported previously.