Superoxide dismutase and catalase cross-linked to polyhemoglobin reduces methemoglobin formation in vitro

Superoxide dismutase (SOD) and catalase (CAT) were evaluated for their ability to reduce methemoglobin formation in chemically cross-linked hemoglobin that was incubated in Tris-HCl buffer. Two parallel studies were carried out using identical experimental conditions with one exception only. This involved the physical state of SOD and CAT in solution such that the enzymes were either cross-linked directly to hemoglobin (hemoglobin-SOD-CAT) or simply incubated with polyhemoglobin. The concentration of SOD and CAT was subsequently varied to examine and compare the effect on the rate of methemoglobin formation between the two experiments. Overall, a comparison between the results clearly indicated that cross-linking SOD and CAT to hemoglobin was more effective at reducing methemoglobin levels than incubating polyhemoglobin with SOD and CAT. More specifically, the incubation experiment would require an enzyme concentration exceeding twice that cross-linked to hemoglobin in order to achieve a similar beneficial effect. However, it was more striking to observe that the presence of SOD and CAT during the cross-linking reaction had markedly reduced the initial formation of methemoglobin. When SOD and CAT were included during the chemical process of generating polyhemoglobin, about 7% methemoglobin was formed in the cross-linked hemoglobin-SOD-CAT product. By comparison, methemoglobin had increased about 20% after the cross-linking reaction when generating polyhemoglobin.