Isolation and recombination of ovine lutropin subunits with complete recovery of biological activity

Abstract

Based on the results obtained in the previous paper (Liu, W.-K., Ascoli, M., and Ward, D. N. (1977) J. Biol. Chem. 252, 5274-5279) we have devised a method that permits the isolation of the subunits of ovine lutropin (oLH) in their "native" form. The hormone was dissociated by guanidine HCl, and the subunits separated by salt precipitation (Sairam, M. R., and Li, C. H. (1974) Arch. Biochem. Biophys. 165, 709-714). The present method differs significantly from that of Sairam and Li in that all operations are carried out at pH 5.0 or higher, a condition we have shown to be crucial to maintain the integrity of the isolated subunits. The yield of subunits was 75 to 85%. The isolated subunits exceed 95% purity (i.e. contamination with the other subunit or intact lutropin is less than 5%), migrate as single bands in sodium dodecyl sulfate-polyacrylamide gels, and show only the NH2-terminal heterogeneity expected from that of the native hormone. Recombination of the subunits can be accomplished with 75 to 85% yield (with respect to mass) and with full recovery of biological activity.