Abstract
Lactoferrin (Lf) and transferrin (Tf) are iron-binding proteins that can bind various metal ions. This study demonstrates the heme-binding activity of bovine Lf and Tf using biotinylated hemin. When both proteins were coated on separate plate wells, each directly bound biotinylated hemin. On the other hand, when biotinylated hemin was immobilized on an avidin-coated plate, soluble native Lf bound to the immobilized biotinylated hemin whereas native Tf did not, suggesting that a conformational change triggered by coating on the plate allows the binding of denatured Tf with hemin. Incubation of Lf with hemin-agarose resulted in negligible binding of Lf with biotinylated hemin. Lf in bovine milk also bound to immobilized biotinylated hemin. These results demonstrate that bovine Lf has specific heme-binding activity, which is different from Tf, suggesting that either Tf lost heme-binding activity during its evolution or that Lf evolved heme-binding activity from its Tf ancestral gene. Additionally, Lf in bovine milk may bind heme directly, but may also bind heme indirectly by interaction with other milk iron- and/or heme-binding proteins.
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10534_2017_75_MOESM1_ESM.docx
Supplementary material Fig. S1 Binding of human and bovine Lf and Tf to hemin-agarose. Human Lf and Tf were purchased from Sigma (St. Louis, MO, USA). Aliquots (1 mL) of Lf or Tf protein (25 µg each) and hemin-agarose beads (HA) or agarose beads (A) in PBS were prepared (net volume of beads per sample: 25 µL each) and incubated at 4°C overnight. The mixture was centrifuged at 14,000×g for 7 min, and the supernatant (S) and pelleted beads were obtained. The pelleted beads were then washed three times with 1 mL of PBS and centrifuged again under the same conditions. Finally, the pelleted beads (B) were subjected to SDS-PAGE together with S. Bovine and human Lf and Tf samples were also separately applied to the gel (2 µg/lane). M represents marker proteins (DOCX 735 kb)
10534_2017_75_MOESM2_ESM.doc
Supplementary material Fig. S2 Binding of bovine milk Lf to hemin agarose. Bovine milk diluted 10-fold with PBS (0.5 mL) was added to 0.5 mL PBS containing a suspension of 50% (v/v) hemin-agarose or agarose (net volume of beads per sample: 25 µl) and the mixture was rotated at 4 °C overnight. The mixture was centrifuged at 1650×g for 7 min at room temperature, then the pelleted beads were washed three times with 1 mL of PBS and centrifuged again under the same conditions. Finally, the pelleted beads were re-suspended in 1.0 mL of ALP-labeled rabbit anti-bovine Lf antibody (250 ng/mL) and incubated (37 °C, 1h). After washing, the pelleted beads were re-suspended with 1 mL of 3 mM disodium p-nitrophenyl phosphate. After incubation at 37 °C, the mixture was centrifuged at 1650×g for 7 min at room temperature and the absorbance of the supernatant was measured at 405 nm. Each value is the mean ± SD of sera obtained from four individuals. * P < 0.01 versus binding examined with agarose beads (DOC 46 kb)
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Saito, N., Iio, T., Yoshikawa, Y. et al. Heme-binding of bovine lactoferrin: the potential presence of a heme-binding capacity in an ancestral transferrin gene. Biometals 31, 131–138 (2018). https://doi.org/10.1007/s10534-017-0075-1
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DOI: https://doi.org/10.1007/s10534-017-0075-1