Partially folded state of the disulfide-reduced N-terminal half-molecule of ovotransferrin as a renaturation intermediate.

A previous report (Hirose, M., Akuta, T., and Takahashi, N. (1989) J. Biol. Chem. 264, 16867-16872) has shown that for the efficient oxidative refolding of disulfide-reduced ovotransferrin, a preincubation under reduced conditions at a low temperature is essential. To study the renaturation pathway, the disulfide-reduced N-terminal half-molecule of ovotransferrin was analyzed by CD spectrum. The reduced protein was found to take, at low temperatures, a partially folded conformation that can be distinguished from both the native and denatured states. The folded protein was in a metastable state with delta GD value of 2.2-2.8 kcal/mol at 6 degrees C. The conformation was variable depending on temperature conditions; its stability was decreased at a lower temperature (1.0-1.2 kcal/mol at 0 degrees C). Subsequent reoxidation at 6 degrees C by oxidized glutathione led efficiently the reduced protein to the correctly renatured form having the iron-binding capacity, indicating that the partially folded state is the immediate precursor to subsequent oxidative refolding.