Protein radicals in the reaction between H₂O₂-activated metmyoglobin and bovine serum albumin

Abstract

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1–2 times stoichiometric excess of H₂O₂ to MMb while a 3–10 times stoichiometric excess of H₂O₂ resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H₂O₂ and BSA were influenced by [H₂O₂] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H₂O₂. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H₂O₂ to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H₂O₂ and the hyper-valent iron-center of H₂O₂-activated MMb.

Keywords:

• Myoglobin
• Ferrylmyoglobin
• Myoglobin ferryl radical
• Protein radicals
• Bovine serum albumin
• ESR
• dityrosine
• protein cross-linking