title: Different stability of N- and C-domain of diferric ovotransferrin in urea and application to the determination of iron distribution between the two domains.
abstract: The study of guanidine-HCl or thermal denaturation of diferric ovotransferrin (Fe2Tf) has revealed a simultaneous unfolding of the two domains of the protein (Ikeda et al. (1985) FEBS Lett. 182, 305-309). In urea denaturation of Fe2Tf, however, two distinct steps of unfolding were observed in the urea concentration range from 4.5 to 9 M at pH 8.0 and 37 degrees C by measuring the residual iron-bound protein (absorbance at 465 nm) and the remaining folded structures (circular dichroism at 222 nm). From a study of urea denaturation of partially iron-saturated Tf whose iron preferentially occupied the N-domain, it was found that the first and the second steps of denaturation corresponded to those of the N-terminal (4.5-6 M urea) and C-terminal domains (over 7 M urea), respectively. The N-domain of Fe2Tf was selectively unfolded in 7 M urea and digested with trypsin to provide an iron-bound C-terminal fragment (42 kDa) in good yield (about 80% of theoretical). The kinetic analysis of the decrease in A465 of Fe2Tf in 9 M urea showed that the N-domain unfolded 3 x 10(2) times faster than the C-domain. With partially iron-saturated Tf, the decrease of A465 in 9 M urea also proceeded in a biphasic manner and the ratio, the decrement in A465 of the rapid phase/the decrement in A465 of the slow phase, gave the value of iron distribution as Fe at the N-site/Fe at the C-site.
authors.affiliation: Department of Chemistry, Faculty of Science, Science University of Tokyo.
authors.forename: K
authors.initials: K
authors.lastname: Nakazato
authors.forename: T
authors.initials: T
authors.lastname: Yamamura
authors.forename: K
authors.initials: K
authors.lastname: Satake
mesh_terms: D001665:Binding Sites; D003207:Conalbumin / Q000031:analogs & derivatives*; D004527:Egg Proteins; D005290:Ferric Compounds; D007501:Iron; D007700:Kinetics; D011489:Protein Denaturation; D014508:Urea
chemical_list: D004527:Egg Proteins; D005290:Ferric Compounds; C057592:diferric ovotransferrin; D003207:Conalbumin; D014508:Urea; D007501:Iron