Oxygen-carrying proteins employed in blood substitute candidates: differences in interactions with a model antioxidant molecule

Glutaraldehyde-polymerized hemoglobin (poly-Hb) has long been explored as a key candidate for blood substitute compositions, to be used in transfusions in order to supplement the oxygen-carrying capacity following severe blood loss. Bovine hemoglobin (bHb) has been the standard choice for such efforts, due to its reasonable availability and to its reduced dependence on organic allosteric effectors. We have recently shown that poly-Hb produced from ovine Hb (poly-oHb) outperforms poly-bHb in in vivo tests employing transfusion after up to 30 % blood loss in animals. This improvement was found to correlate with an increased resistance of ovine hemoglobin (oHb) and of poly-oHb towards oxidative and nitrosative stress agents in vitro. The molecular bases for these differences in reactivity offer an interesting challenge, given the high sequence homology between vertebrate hemoglobins. Reported here is an investigation of these molecular bases using different spectroscopic (fluorescence, resonance Raman, NMR, EPR) and computational (molecular docking) methods to assess the interaction with a convenient probe ligand representative of the class of natural antioxidants, caffeic acid. Fluorescence experiments reveal that ovine Hb fluorescence saturates above 25 μM caffeic acid and indicating full occupancy of fluorescence-responsive binding sites, while resonance Raman and NMR data indicate signals for the heme and indicate the differences between the types of Hbs and the antioxidant binding behavior after polymerization. Computational docking corroborated the spectroscopic data by identifying aromatic residues and distinct affinity patterns for caffeate. The results show that structural differences in oHb may explain a higher redox stability.