Formation of Long-Lived Protein Radicals in the Reaction Between H 2O 2-Activated Metmyoglobin and Other Proteins

Abstract

Free radicals formed during the reaction of H 2O 2 and metmyoglobin in the presence of bovine serum albumin (BSA) were investigated using freeze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0–300 μM) resulted in drastic changes in the characteristic freeze quench ESR signal of H 2O 2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 μM H 2O 2, 100 μM metmyoglobin, and 200 μM BSA have half-lives of approximately 13 min at 25°C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25°C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, γ-globulin or β-lactoglobulin also resulted in formation of long-lived free radical species (half-lives: 13–18 min); however, the ability to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > γ-globulin > β-lactoglobulin. The spin-trap α-phenyl- tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H 2O 2, and BSA. Transient radical species that could be proposed as intermediates in the formation of the long-lived protein radicals detected by freeze-quench ESR. Dityrosine was formed in the reaction between MMb, H 2O 2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H 2O 2-activated myoglobin and other proteins have major consequences on future interpretations of the significance of the perferryl protein radical in biological systems where proteins are abundant.