Renaturation of ovotransferrin under two-step conditions allowing primary folding of the fully reduced form and the subsequent regeneration of the intramolecular disulfides

Abstract

A two-step procedure was found to be useful for the efficient refolding of a complex protein, ovotransferrin. In the first step, the reduced and denatured form of the protein was incubated at a low temperature in a nondenaturing buffer containing reduced glutathione; in the second step, the reduced form was reoxidized at a higher temperature in the presence of oxidized glutathione. Under these conditions, the fully reduced forms of ovotransferrin and its half-molecules were almost quantitatively reoxidized to regain iron-binding abilities and conformations, very similar to the native form. The circular dichroism spectra revealed that at low temperatures the fully reduced forms have partially folded conformations, which are fluctuating like "molten globule" states. The reoxidization kinetics compared between whole ovotransferrin and the two half-molecules supported independent refolding of the N- and C-terminal domains.