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J. Biochem, 1988, Vol. 103, No. 5 823-828
© 1988 Japanese Biochemical Society

research-article

Different Stability of N- and C-Domain of Diferric Ovotransferrin in Urea and Application to the Determination of Iron Distribution between the Two Domains1

Katsuyoshi Nakazato, Faculty of Science2, Takaki Yamamura, Faculty of Science3 and Kazuo Satake, Faculty of Science

Department of Chemistry,Science University of Tokyo Shinjuku-ku, Tokyo 162

2To whom correspondence should be addressed at: Niigata College of Pharmacy, Department of Chemistry, Niigata, Niigata 950-21.

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°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 × 102 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.

1This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

3Present address: Internal Res. Lab., CIBA-GEIGY Japan Limited, Takarazuka, Hyogo 665.


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