Photon Factory Activity Report 2014 #32 (2015) B

BL-10C 2014R-36

Association Mechanism of Lactoferrin with Multiferric Ions
Tomohiro Yamaguchi1, Shohei Seino1, Hiroshi Kawakami2, Masaru Tanokura3 and Masaki Kojima1*
1School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
2
Faculty of Home Economics, Kyoritsu Women's University, Tokyo 101-0003, Japan

1 Introduction
Bovine Lactoferrin (Lf) from milk is an iron binding
protein, and its biological function such as antibacterialactivity, regulation of iron absorption, and regulation ofthe production of macropharges and granulocystes has
been well studied. Lf is folded in two lobes, each ofwhich contains a binding site for an Fe3+ ion [1].
On the other hand, Lf can also bind more Fe3+ ions(about 70 ions per protein molecule) at other sites in thepresence of bicarbonate. Various physicochemical
intestigations for this form of Lf (70FeLf) have revealed
that 70FeLf is more stable than intact Lf and that 70FeLf
contains 15 or 16 protomers whereas native Lf is amonomer [2]. It is also suggested that the quaternary
structure of 70FeLf can be stabilized throgh theconjunction of CO32- with exesssive Fe3+ ions, although
the detailed mechanism of the association of Lf withmultiferric ions is still unclear.
In this study, we investigated the architecture of70FeLf by small-angle X-ray scattering (SAXS).
2 Experiment
Native Lf and 70FeLf were purified in the samemanner as previously reported [2]. The protein solution
was prepared at various concentrations from 0.5 to 2
mg/mL at pH 1.0 – 6.0.
All the experiments were performed at BL10C andBL15A. SAXS data were measured at a sample-todetector length of 1 m and at 20OC with PILATUS
detector. The exposure time was 60 s in one measurement.
The sample in the cuvette was exchanged every threetimes. Bovie serum albumin was also measured for
caribration.
Guinier analyses and singular value decomposition(SVD) were performed with our in-house program.
3 Results and Discussion
SAXS data of 70FeLf showed multidisperse profiles ina wide range of pHs, whereas native Lf was shown as
monodisperse, which was confirmed by SVD analysis. Itwas suggested that the system may consist of randomly
aggregated form and Fe3+-bound complex. Therefore we
performed Guinier analysis with two-exponential fitting,and characterized structural properties of the latter majorcomponent.
The radius of gyration (Rg) of Fe-Lf complex wasranged from 40 to 50 angstrom, and hardly changed atvarious pHs. On the other hand, molecular weight (Mw)estimated from forward scattering was changed aroundpH 3, which suggested the contribution of CO32- ion upon
the formation of Fe-Lf complex.
Table 1: pH profile of 70FeLfpH136Rg (Å)
Mw (kDa)49.2

45.7

43.8

439

525543

Based on these results, we are now constructing themolecular model of the complex that can elucidate theassociation mechanism of Lf and multiferric ions.

Acknowledgement
This work was supported by Platform for Drug
Discovery, Informatics, and Structural Life Scieence from
the Ministry of Education, Culture, Sports, Science andTechnology, Japan.
References[1] S. Moore et al., J. Mol. Biol. 274, 222 (1997).
[2] F. Hu et al., International Dairy Journal 18, 1051
(2008).
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* mkojima@toyaku.ac.jp