Synthesis and characterization of hemoglobin conjugates with antioxidant enzymes via poly(ethylene glycol) cross-linker (Hb-SOD-CAT) for protection from free radical stress

Hemoglobin (Hb) conjugated with the antioxidant enzymes (SOD and CAT), by employing dicarboxymethylated poly(ethylene glycol), was designed for protection of hemoglobin against free radicals. In this study, the conjugation process was confirmed by employing SDS-PAGE and SEC techniques. The average molecular weight of the conjugates was estimated to be around 1000 kDa. The enzymatic activities of the SOD and CAT in the conjugates (Hb-SOD-CAT) after conjugation were found to retain greater than 70% and 90% of the original bioactivity. Results show that antioxidant enzymes helped minimize methemoglobin (non-carrier of oxygen) formation during the conjugation process and also during storage at 4 °C over a period of 1 month. In summary, the optimized (1:10 Hb/PEG) crosslinked conjugates with antioxidant enzymes showed protective properties from severe free radical stresses when incubated with hydrogen peroxide (0.1 and 1 mM) and xanthine (1 mM)/xanthine oxidase (10 and 20 mU/ml) system.