Abstract
A procedure is described for the purification of pituitary gonadotropin from the salmon ( Oncorhynchus tshawytscha) by means of ethanol extraction, gel filtration on Sephadex G-100 (preparation SG-G100) and ion exchange chromatography on DEAE-cellulose; (preparations SG-DEAE-1 to 7). Bioassays were carried out using either the goldfish spermiation response or the augmentation of radiophosphate uptake by chick testis. In the goldfish assay, 1 mg SG-G100 and SG-DEAE-2 were equivalent to approximately 2150 IU HCG and 10,000 IU HCG, respectively. In the chick assay, SG-G100 had a potency of 0.10 × NIH-LH-S16. The sedimentation coefficients s 20 w for SG-DEAE-3 and SG-G100 were determined by ultracentrifugation to be 2.57 and 2.65, respectively. Molecular weights (M) calculated using the Svedberg equation for SG-DEAE-3 and SG-G100 were 28,524 and 29,411, respectively. At pH 1.4, s 20 w for SG-G100 was 1.38 and M = 12,805. Direct determination of M by gel filtration using nonglycoproteins as standards gave an M value for SG-G100 of 40,000. Stokes radius for SG-G100 was determined by gel filtration to be 26.5 Å and a diffusion coefficient of 8.9 × 10 −7 cm 2 sec −1 was calculated.
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