title: Formation of long-lived protein radicals in the reaction between H2O2-activated metmyoglobin and other proteins.
abstract: Free radicals formed during the reaction of H2O2 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 microM) resulted in drastic changes in the characteristic freeze quench ESR signal of H2O2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 microM H2O2, 100 microM metmyoglobin, and 200 microM BSA have half-lives of approximately 13 min at 25 degrees C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25 degrees C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, gamma-globulin or beta-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 > gamma-globulin > beta-lactoglobulin. The spin-trap alpha-phenyl-tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H2O2, 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, H2O2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H2O2-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.
authors.affiliation: Danish Institute of Animal Science, Department of Product Quality, Research Center Foulum, Tjele.
authors.forename: H
authors.initials: H
authors.lastname: Ostdal
authors.forename: L H
authors.initials: LH
authors.lastname: Skibsted
authors.forename: H J
authors.initials: HJ
authors.lastname: Andersen
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D006207:Half-Life; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry; D008786:Metmyoglobin / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D005719:gamma-Globulins / Q000737:chemistry
chemical_list: D016166:Free Radical Scavengers; D005609:Free Radicals; D007782:Lactoglobulins; D012709:Serum Albumin; D005719:gamma-Globulins; D008786:Metmyoglobin; D006861:Hydrogen Peroxide