Surface-induced unfolding of human lactoferrin

Jian R. Lu, Shiamalee Perumal, Xiubo Zhao, Fausto Miano, Vincenzo Enea, Richard R. Heenan, Jeff Penfold

    Research output: Contribution to journalArticlepeer-review


    We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 Å on the air side and a bottom diffuse layer of some 50 Å into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm-3, close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 × 10-8 mol m-2 in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 × 10-8 mol m-2. Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 Å and a length of 105 Å in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 Å and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption. © 2005 American Chemical Society.
    Original languageEnglish
    Pages (from-to)3354-3361
    Number of pages7
    Issue number8
    Publication statusPublished - 12 Apr 2005


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