An interchain disulfide dimer of human growth hormone.

Human pituitary glands contained a form of growth hormone with a molecular weight of 45,000 that did not dissociate in 8 M urea or 1% sodium dodecyl sulfate. The material was isolated by a combination of ion exchange chromatography on DEAE-cellulose in a urea-containing buffer and gel filtration on Sephadex. Upon reduction with 2-mercaptoethanol or oxidation with performic acid prior to electrophoresis in sodium dodecyl sulfate, the 45,000-dalton protein was converted into material with a molecular weight of 22,000. The 45,000-dalton form had a low growth-promoting activity, 0.14 IUlmg, and, after reduction and Cysteinecarbamidomethylation, the potency of the material increased to 0.55 IU/mg. The lactogenic activity of the 45,000dalton form was 1.7 IU/mg and this remained essentially unchanged upon reduction and alkylation. In a radioimmunoassay the 45,000-dalton protein was about 50% and the 22,000-dalton Cysteine-carbamidomethylated material, about 35% as effective as human growth hormone in displacing labeled growth hormone. Phenylalanine was the NH,-terminal residue for the 45,000-dalton form and for the Cysteine-carbamidomethylated 22,000-dalton material. Sequence analysis by the Edman procedure indicated the first 6 residues of these materials to be the same as those found in human growth hormone. Phenylalanine was the COOH-terminal amino acid and glytine the penultimate residue. Formation of the 45,000-dalton protein was not detected after permitting monomeric growth hormone to stand at pH 9 for 1 week. Since all purification steps of the 45,000-dalton material were carried out at a pH of between 6.5 and 8.3, its formation by disulfide interchange seemed unlikely. We concluded that the 45,000-dalton protein was a dimer of growth hormone formed by interchain disulfide bonding. In addition to the disulfide dimer, another dimeric form of human growth hormone was detected which was resistant to reduction by 2-mercaptoethanol. The substance was only about 15% as active as human growth hormone in a radioim-