Purpose: To investigate the protective effect of cross-linked Hb, SOD, and CAT using the conjugating agent poly (ethylene glycol) (PEG, 2K) on rat insulinoma cells (RINm5F).

Methods: Freshly isolated Bovine Hemoglobin (Hb) from red blood cells was conjugated with two antioxidant enzymes (SOD and CAT) using water soluble polymer PEG (2K). The conjugation ratio of Hb to PEG was fixed at 1:15 mole:mole and CAT and SOD were added in the ratio of 1:20 Enzyme Units (15000U and 300000U). The conjugated formulations were evaluated by visible spectroscopy for methemoglobin formation, and Size Exclusion Chromatography (SEC) to determine size. The biological activities of the enzymes were confirmed by enzymatic assays. The final conjugates were also evaluated for their anti-oxidant effects by treating cells with two different insulin secreting cell-specific damaging oxidants (Hydrogen Peroxide and Superoxide anions). Cell viability was determined using MTT assays.

Results: The visible absorption spectrum of the formulations did not show any formation of methemoglobin (630nm) after the conjugation process. SEC analysis showed that the molecular conjugates were larger than 1000 kDa. When rat insulinoma cells (RIN5mF) were treated with 1mM Hydrogen peroxide (H202), cells were protected only by formulations containing Catalase. When cells were treated with superoxide anions generated using the HypoXanthine/ Xanthine Oxidase system (0.1mM/2mU/mL) a protective effect was observed when formulations contained SOD. Cells were protected from the combination of oxidants only when they were treated with formulations containing both SOD and CAT.

Conclusions: A novel anti-oxidant formulation for the protection of insulinoma cells was created. The final conjugate showed a protective effect following oxidant challenges (peroxide and superoxide anion). These preliminary results indicate that Hb-SOD-CAT conjugates can act as a suitable free radical protective agent, thus protecting insulinoma cells from both hypoxic and free radical stresses. Further experiments on these cells combining hypoxia and oxidative challenges are in progress. The conjugates will be applied to protect transplanted encapsulated pancreatic islets.