Purification and properties of follicle-stimulating and luteinizing hormones from horse pituitary glands

A method is described for the purification of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from horse pituitary glands. The glands were extracted twice in 40% ethanol, and the active protein was recovered by acetone precipitation. Inactive protein was removed by dialysis of the acetone precipitate in dilute NaCl, precipitation with HPO 3, gel filtration on Sephadex G-100, and ion-exchange chromatography on CM-Sephadex. FSH and LH activities were separated by preparative disc electrophoresis at pH 8.9. Additional purification was accomplished by gel filtration of FSH on Sephadex G-75 and LH on Sephadex G-100. The FSH, containing 90 units per mg of NIH-FSH-S1 and 0.16 units per mg of NIH-LH-S1, was obtained in a yield of 26 mg per kg of fresh tissue. It was homogeneous by disc electrophoresis at pH 8.9 and pH 7.5, Ouchterlony double diffusion, immunoelectrophoresis, and sedimentation equilibrium ultracentrifugation. After disc electrophoresis at pH 4.3, two zones were apparent, but FSH activity was lost. The molecular weight of the horse FSH was 33,200 by ultracentrifugation and 47,900 by gel filtration. An isoelectric point of pH 4.1 was determined by electrofocusing. The LH, containing 5.3 units per mg of NIH-LH-S1 by the ovarian ascorbic acid-depletion assay and 34 units per mg by the ventral prostate-weight assay, showed 10% FSH activity. Several components were seen after Ouchterlony double diffusion and immunoelectrophoresis. Heterogeneity was apparent by disc electrophoresis, and bioassay of gel slices indicated a number of LH components and an FSH component with a different electrophoretic mobility than the major horse FSH fraction. Four LH components with isoelectric points at pH 4.5, 5.9, 6.6, and 7.3 were shown by electrofocusing. An FSH component with an isoelectric point at pH 4.8 was also present. The molecular weight of the horse LH preparation was 44,500 by ultracentrifugation and 63,800 by gel filtration.