Pregnant Mare Serum Gonadotropin

Procedures have been developed for the purification of pregnant mare serum gonadotropin (PMSG) and its a and jl subunits. The procedure for the hormone purification involves three steps of column chromatography on Sephadex G-100, DEAE-Sephadex A-50, and hydroxyapatite. The preparation of subunits involves the dissociation of PMSG with 10 M urea followed by their separation by chromatography on DEAE-Sephadex and Sephadex G-100. The hormone and subunit preparations were found homogeneous by electrophoresis in polyacrylamide gel with or without sodium dodecyl sulfate and by immunodiffusion. The hormone had an activity of 13,740 I.U./mg as determined by in viva and in vitro bioassays and receptor binding assays. The subunits did not show any significant activity by the receptor binding assay. The molecular weights of PMSG, PMSG-e, and PMSG-P, determined from Ferguson plots (Ferguson, K. A. (1964) Metabolism 13, 9851002) using glycoproteins as molecular weight markers, were 64,030,43,720 and 16,960, respectively. The amino acid and carbohydrate compositions of the hormone and the subunits have been determined. The carbohydrate content of the hormone was 41.7% and the (Y and jl subunits contained 20.6 and 45.6% carbohydrate, respectively (uncorrected for moisture content of protein). The carbohydrate moiety of the hormone is made up of L-fucose (0.6 to 0.9%), D-mannose (2.0 to 2.3%), Dgalactose (10.6 to 12%), N-acetylglucosamine (9.0 to 10.5%), N-acetylgalactosamine (3.0 to 3.5%), and sialic acid (12.0 to 14.0%). The purified PMSG was found to be three times as active as ovine lutropin (LH, luteinizing hormone) (2.3 NIH-LH-SI units/mg) and 2/3 as active as ovine follitropin (FSH, follicle-stimulating hormone) (115.3 NIHFSH-SI units/mg). FSH activity was determined by the Steelman-Pohley assay (Steelman, S., and Pohley, F. (1953) Endocrinology 53, 604-616) and the LH activity was measured by ascorbic acid depletion assay. As determined by binding assay, the individual subunits upon recombination recovered 27.2% of the LH activity and 62.5% of the FSH activity. Immunologically, PMSGa and PMSG-j? cross-reacted with anti-PMSG as found by radioimmunoassay. While human chorionic gonadotropin (hCG) and human-luteinizing hormone (hLH) competed with ‘“‘I-PMSG in the PMSG receptor binding assay, they showed little or no cross-reactivity in the