Hemoglobin (10 µmol/L) was oxidized with H2O2 (40 µmol/L) at room temperature for 30 min. To assess the effect of haptoglobin on the fragmentation of hemoglobin, hemoglobin was incubated with haptoglobin (Hp 1-1, 330-12, Lee BioSolutions, 10 µmol/L) and then exposed to H2O2 (40 µmol/L) at room temperature. The oxidized products were concentrated with an Amicon Ultra-0.5 centrifugal filter unit with an Ultracel-10 membrane (UFC501096, Merck KGaA, Darmstadt, Germany). The concentrated fraction (containing 30 µg of protein) was subjected to SDS-PAGE (NW04120BOX, NuPAGE Bis-Tris Precast Gel, Thermo Scientific).
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