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Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

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Abstract

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.

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  1. Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Towada City, Aomori, 034-8628, Japan

    Koichi Orino

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  1. Koichi Orino
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Correspondence to Koichi Orino.

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Fig. S1

The binding of human fibrinogen-Sepharose 4B with Zn ion (A) and the effect of Zn ions on the binding of human fibrinogen with Zn-PPIX (B). A) One ml aliquot of T buffer containing 20 μM ZnSO4 and 50 μl of human fibrinogen-Sepharose 4B beads (solid bar) or Sepharose 4B beads (open bar) were incubated at room temperature for 30 min. After incubation, the mixture was centrifuged at 14,000×g for 5 min. The concentration of Zn in the supernatant was measured using a Metallo Assay Zinc LS kit (AKJ Global Technology Co., Ltd., Chiba (Chiba, Chuo, Japan) to determine Zn recovery. The zinc concentration was normalized to the control (100 %) in the absence of beads. Each value is the mean ± SD of four replicates. *P < 0.001, compared with the control beads. (B) ZnSO4 was added to a final concentration of 20 μM to an aliquot (1 ml) containing 10 μM Zn-PPIX and human fibrinogen-Sepharose 4B (solid bar) or Sepharose 4B (open bar) beads (net volume of beads per sample: 50 μl) in T buffer containing 10 μM NaOH. The mixture was incubated at room temperature for 30 min followed by centrifugation at 14,000×g for 5 min. The supernatant was spectrophotometrically measured at 390 nm. The binding of Zn-PPIX to fibrinogen was normalized to the control (100 %) in the absence of beads. Each value is the mean ± SD of six replicates. *P < 0.001, compared with the control beads.

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Orino, K. Functional binding analysis of human fibrinogen as an iron- and heme-binding protein. Biometals 26, 789–794 (2013). https://doi.org/10.1007/s10534-013-9657-8

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  • DOI: https://doi.org/10.1007/s10534-013-9657-8

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