Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+

Abstract

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO •) generating systems of xanthine oxidase (XO) + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA−Fe 3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO •. The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA−Fe 3+ but not to the ascorbate + EDTA−Fe 3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H 2O 2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA−Fe 3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA−Fe 3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA−Fe 3+ system but not to the XO + EDTA−Fe 3+ system.