STRUCTURAL CHARACTERISTICS OF THE DISULFIDE-REDUCED OVOTRANSFERRIN N-LOBE ANALYZED BY PROTEIN FRAGMENTATION

The isolated N-lobe (1-332) and C-lobe (342-686) of ovotransferrin wer e each found to form an opaque gel by incubation with 70 mM 2-mercapto ethanol at 37 degrees C for 24 h, suggesting that a protein conformati onal change due to extensive disulfide reduction was the initial mecha nism prior to subsequent intermolecular protein aggregation forming ge l networks. To investigate the conformational state of an extensively disulfide-reduced protein, the structural characteristics of a two-dis ulfide protein form that had been shown to be produced by extensive di sulfide reduction of the N-lobe [H. Yamashita, T. Nakatsuka, and M. Hi rose, J. Biol. Chem., 270, 2980-29812 (1995)] were analyzed, as a mode l protein, by the protein fragmentation approach, When the two-disulfi de form was proteolyzed with chymotrypsin, a protease-resistant 8-kDa fragment was produced. This fragment was purified by ion-exchange colu mn chromatography, and chemical analyses and matrix-assisted laser des orption ionization time-of-flight mass spectrometry clearly showed tha t the 8-kDa fragment comprised Ala1-Tyr72 of ovotransferrin. The fragm ent was found by amino acid analysis to retain the two intra-chain dis ulfide bonds intact. Far-UV CD spectrum analyses revealed that the 8-k Da fragment retained a native-like folded conformation. These data are consistent with the view that the N-terminal segment, Ala1-Tyr72, ass umed a local native-like conformation in the two-disulfide form of the ovotransferrin N-lobe.