Purification and Subfractionation of Bovine Thyrotropin and Lutropin Using Radioimmunoassays for Evaluation of the Purification Processes1

Abstract

Procedures for the group separation of bovine pituitary glycoprotein hormones, i.e., thyrotropin, lutropin, and follitropin, and for the purification and subfractionation of the first two hormones were studied. Radioimmunoassay systems specific to bovine thyrotropin and lutropin have been developed and used to evaluate these purification processes. Pituitary glycoprotein hormones were fractionally extracted with aqueous ethanol from 400 g of bovine lyophilized pituitary powder by the percolation method and purified by bioaffinity chromatog-raphy on a column of concanavalin A-Sepharose; more than 500-fold purification was achieved within 4-5 days and the estimated overall yield was close to 90% of the total extractable hormones. A mixture of thyrotropin and lutropin was obtained efficiently from this glycoprotein hormone concentrate by CM-cellulose chromatography and further fractionated by DEAE-cellulose chromatography. Although most of the lutropin was separated from thyrotropin on this chromatography, thyrotropin was always accompanied by a substantial amount of lutropin. On isoelectric focusing of the thyrotropin-containing fraction thus obtained, five peaks of immunologic thyrotropin with isoelectric points at pH 7.20, 7.90, 8.32, 8.60, and 8.95 were obtained; contaminating lutropin was also distributed over this pH range. Thyrotropin components completely free of lutropin could be obtained by the use of affinity chromatography on a column of immobilized anti-bovine lutropin antibodies. On the other hand, four components of bovine lutropin, completely or almost completely free of thyrotropin, were purified by isoelectric focusing of two lutropin fractions obtained by DEAE-celluIose chromatography. Their isoelectric points were distributed over a pH range of 8.2-9.0.