Oxidative Protection of Hemoglobin and Hemerythrin by Cross- Linking with a Nonheme Iron Peroxidase: Potentially Improved Oxygen Carriers for Use in Blood Substitutes Denisa Hathazi, † Augustin C. Mot, † Anetta Vaida, † Florina Scurtu, † Iulia Lupan, ‡ Eva Fischer-Fodor, § Grigore Damian, ∥ Donald M. Kurtz, Jr., ⊥ and Radu Silaghi-Dumitrescu* ,† † Faculty of Chemistry and Chemical Engineering, “Babes-Bolyai” University, 11 Arany Janos St., Cluj-Napoca, Romania ‡ Institute of Interdisciplinary Research in Bio-Nanosciences, Molecular Biology Center, 42 Treboniu Laurean St., Cluj-Napoca, 400271-Romania ∥ Faculty of Physics “Babes-Bolyai” University, 1 Kogalniceanu St., Cluj-Napoca, Romania § Ion Chiricuta Cancer Institute - Comprehensive Cancer Center, 34-36 Republicii St., Cluj Napoca, Romania ⊥ Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249-0698, United States * S Supporting Information ABSTRACT: The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times. ■ INTRODUCTION The use of hemoglobin (Hb)-based artificial oxygen carriers in blood substitutes has been described and examined exten- sively. 1,2 However, their applicability so far has been limited due to toxicity issues, at least some of which can be linked to oxidative stress. Strategies for limiting this toxicity have included the addition of antioxidant small molecules or enzymes to chemically derivatized or encapsulated particles of Hb. 1,3,4 The nonheme iron oxygen carrying protein, hemerythrin (Hr) has recently been proposed as an alternative to Hb in blood substitutes. 5 Hr is an oxygen transport and storage protein found in marine invertebrates that employs a nonheme diiron site Fe(II)−Fe(II) to reversibly bind dioxygen. Hr has some potential advantages over Hb for a blood substitute, including a higher molecular weight (108 kDa vs 64 kDa Hb), which can lead to lower extravasation or elimination through the kidney (hypertension and vasoconstriction are inversely correlated with the size of the blood substitute protein), 4 lower reactivity toward hydrogen peroxide, nitric oxide, and nitrite and a remarkably lower tendency to generate toxic free radicals in such reactions. 5−7 In the absence of the enzymatic system in red blood cells, Hb undergoes a series of reactions generating potentially toxic reactive oxygen species such as hydrogen peroxide or superoxide. As a strategy to scavenge the reactive oxygen species generated by Hb, PolyHb-superoxide dismutase-catalase was designed so that the blood substitute contains copoly- merized antioxidant enzymes. 3 We propose to combine alternative antioxidant enzymes with either Hb or Hr. One of these alternative antioxidant enzymes, rubrerythrin (Rbr) is a nonheme iron protein with peroxidase (hydrogen peroxide reductase) function. 8 Unlike heme-based peroxidases or catalases, the reduced (all-ferrous) Rbr reduces hydrogen peroxide to water at a diiron site, which does not involve high-valent iron (Fe IV or Fe V ) or free radicals (porphyrin- or protein-based). Moreover, the K m for hydrogen peroxide is 2 orders of magnitude lower for Rbr compared to those of peroxidases and catalases. 9−12 The oxidized (all-ferric) Rbr also shows a low level of aromatic substrate peroxidase Received: March 19, 2014 Revised: April 9, 2014 Published: April 9, 2014 Article pubs.acs.org/Biomac © 2014 American Chemical Society 1920 dx.doi.org/10.1021/bm5004256 | Biomacromolecules 2014, 15, 1920−1927