Figure 2. Dose-dependent oxidation of C_18:2,n−6 by added Mb and H₂O₂.

Increasing yield of 9(R/S)-HODE (•), 13(R/S)-HODE (▴) (measured at 234 nm) and total oxoODE (▪) (measured at 270 nm) was determined in mixtures containing C_18:2,n−6 (1 mM) and an increasing dose of wild-type Mb (A), Y103F Mb (B) each treated with H₂O₂ (Mb/H₂O₂ ratio, 5 mol/mol) or corresponding concentrations of H₂O₂ alone (C). All reaction mixtures were incubated for 15 min at 37 °C, worked-up as outlined in the Experimental section and analysed for oxidation products with HPLC. Results are means±S.D. of three independent analyses. Where error bars are not shown, the symbol is larger than the error. Inset in (C) shows the electronic absorbance spectrum for wild-type Mb (2.5 μM, solid line) immediately after addition of purified C_18:2,n−6 (100 μM, dotted line) and addition of H₂O₂ (12.5 μM, broken line) to the mixture. Note, solid and dotted lines overlap with a small decrease in peak intensity detected at A₄₀₉. A shift in Soret maxima (A₄₀₉ to A₄₁₉), indicated by the arrows, together with a concomitant decrease in peak intensity, is consistent with the formation of ferryl Mb upon addition of H₂O₂. DTPA (200 μM) was included in all reaction mixtures. au, arbitrary units.