The oligosaccharide on alpha-subunit loop 2 (alpha2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F). Re-assembly of human choriogonadotropin, human follitropin, and bovine lutropin occurred rapidly and efficiently following removal of the alpha2 oligosaccharide by PNGase F. Consequently, virtually all heterodimers formed in the presence of this enzyme lacked this oligosaccharide. These findings support the notion that heterodimer assembly in vitro occurs by a threading mechanism that is impeded by the presence of the alpha2 oligosaccharide. This procedure should facilitate the study of glycoprotein hormone structure and function. (C) 2003 Elsevier Science (USA). All rights reserved.