Chemical and Biological Properties of Gonadotropin-Releasing Hormone Synthesized by the Solid-Phase Method*

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Abstract
The decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, corresponding to the amino acid sequence of porcine gonadotropin-releasing hormone (Gn-RH), was synthesized by the solid-phase method. The protected product was purified by chromatography on two silica-gel columns and crystallization from hot methanol. Following hydrogen fluoride deblocking and anion-exchange, the peptide was purified by gel-filtration through Sephadex G-25 followed by Sephadex G-15. This product was analyzed by optical rotation, elemental analysis, thin-layer electrophoresis chromatography, amino acid analysis, and digestion by α-chymotrypsin, leucine amino-peptidase, and carboxypeptidase-A. By these criteria the synthetic material was determined to be optically and chemically, greater than 96% pure. In vitro releasing activity of the peptide was determined by incubation of hemisected anterior pituitaries from male rats in a complete tissue culture media. FSH release was measured by specific bioassay, as well as by radioimmunoassay. LH release was measured by radioimmunoassay for rat LH. Media FSH was increased 3- and 6-fold above control levels by 1 and 5 ng of peptide, respectively. LH was increased 4- to 7-fold in the same media by the peptide. In vivo, following intravenous injection of peptide, a highly significant increase in serum LH, but not FSH, as measured by specific radioimmunoassay, was observed in the ovariectomized, estrogen-progesterone-treated rat. In the lateanestrous ewe, maximum serum LH and FSH occurred within 90 min of Gn-RH injection at 100-fold and 165% of baseline, respectively. The ability of the peptide to induce ovulation was determined in mature New Zealand rabbits. Twenty to 24 hr after a single intravenous injection of peptide, the rabbits were sacrificed and the number of ovulations, unruptured follicles, and recovered ova were used as response criteria. Fifty percent of the rabbits ovulated at a dose of 1.25 μg compared to 100 percent at a dose of 2.5 μg synthetic Gn-RH. (Endocrinology93: 96, 1973)

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