Figures & data
Figure 1. UV-vis spectra of hemoglobin derivatized with oATP in the presence of various antioxidants; also shown for reference are spectra of underivatized oxy and met Hb. The spectra of derivatized samples are obtained by diluting equal amounts of each sample into 1 mL PBS. The concentrations of the oxy, met, and hemichrome forms (in this order, shown in parentheses, in micromolar) in each sample, calculated from Winterbourn equations as detailed in the Methods section, are as follows: 10 mM oATP (0.9, 7.0, 0.3), ascorbate (7.2, 2.9, 0.1), thiosulfate (6.8, 0.3, 0.0), glucose (0.0, 7.3, 0.9), 1 μM BSA (0.0, 6.6, 0.4), 10 μM BSA (0.4, 7.2, 0.4), 100 μM BSA (1.2, 4.9, 0.3), 500 μM BSA (5.8, 2.2, 0.0), 1 mM BSA (4.9, 1.2, 0.0), 2 mM BSA (6.7, 0.7, 0.0).
Figure 2. SDS-PAGE of oATP-derivatized hemoglobin in the presence of various antioxidants: 1-10oATP, 2- + ascorbate, 3- + thiosulfate, 4- + glucose, 5- + 1 μM BSA, 6- + 10 μM BSA, 7- + 100 μM BSA, 8- + 500 μM BSA, 9- + 1 mM BSA, 10- + 2 mM BSA, 11– pure Hb.
Figure 3. Gel filtration chromatograms for selected samples.
Table 1. Autooxidation rates (expressed in percentage increases relative to native hemoglobin), P50 values (also expressed as percentage increases), and Hill coefficients (n)for oATP-produced BSA-Hb copolymers.
Figure 4. The reaction of derivatized hemoglobins with H2O2. Conditions: room temperature, 10µM Hb (native or subjected to derivatization with oATP and BSA at concentrations indicated), 100 µM H2O2, PBS 7.4.
Figure 5. EPR spectra of 200 μM oATP-Hb and oATP-Hb-BSA copolymers treated with 500 μM H2O2 in PBS 7.4 and frozen at 20 s after mixing. Insert shows free radicals region of the samples (3300-3400 G, with intensities differing from sample to sample); the 1000-1500 G region shows small signals due to ferric iron, with similar intensities in all samples.
