Superoxide dismutase (SOD) and Catalase (CAT) play a crucial role as the first line of defense antioxidant enzymes in a living cell. These enzymes neutralize the superoxide anion from the autooxidation of oxyhemoglobin (Oxy-Hb) and convert hydrogen peroxides into water and molecular oxygen. In this study, we fabricated hemoglobin submicron particles (HbMPs) using the Coprecipitation Crosslinking Dissolution (CCD) technique and incorporating first-line antioxidant enzymes (CAT, SOD) and second-line antioxidant (ascorbic acid, Vit. C) to investigate a protective effect of modified HbMPs via cyclically oxygenation and deoxygenation. Thereafter, the total hemoglobin (Hb) content and Oxy-Hb content to HbMPs were determined. The results revealed that the HbMPs have a protective effect against oxidation from hydrogen peroxide and potentially neutralizing hydrogen peroxide to water over 16 times exposure cycles. No significant differences in total Hb content were found between normal HbMPs and enzyme-modified HbMPs in the absence of Vit. C. The Oxy-Hb of CAT-HbMPs showed significantly higher values than normal HbMPs. The functional Hb of normal HbMPs and enzyme-modified HbMPs was increased by 60-77% after a short time Vit. C (1:25) exposure. The co-immobilization of CAT and SOD in hemoglobin particles (CAT-SOD-HbMPs) in the presence of Vit. C provides protective effects against oxidation in cyclic Oxygenation and Deoxygenation and shows the lowest reduction of functional Hb. Our studies show that the CCD technique-modified HbMPs containing antioxidant enzymes and a reducing agent (ascorbic acid) demonstrate enhanced Hb functionality, providing protective effects and stability under oxidative conditions.
