Oxidation of DBBF-Hb by low levels of enzymatically generated H₂O₂.

(A) Reaction mixtures were prepared containing 50 μM DBBF-HbFe²⁺, DBBF-HbFe³⁺, or DBBF-HbFe³⁺CN in HBSS, pH 7.4, 37°C. Representative visible spectra (450-700 nm) are shown before and 2 hours after the addition of 10 mU/mL GOX. After 2 hours, the spectra obtained with DBBF-HbFe²⁺ and DBBF-HbFe³⁺ were similar and reflected the formation of the Fe⁴⁺ intermediate. Arrows indicate the direction of change of the spectra. The characteristic spectrum of DBBF-HbFe³⁺CN was unchanged. (B) Time-dependent changes in the redox states after GOX addition to DBBF-HbFe²⁺. Visible absorbance spectra were collected every 5 minutes for 2 hours, and the oxidation states were calculated using the Winterbourn method. (C) Ferryl hemoglobin formation after the addition of 500 μM H₂O₂ or 10 mU/mL GOX to medium containing DBBF-Hb under culture conditions. Ferryl hemoglobin was measured using the sodium sulfide method as described in “Materials and methods.” Each point represents the mean ± SE for 4 to 5 samples.