FORMATION OF LONG-LIVED PROTEIN RADICALS IN THE REACTION BETWEEN H2O2-ACTIVATED METMYOGLOBIN AND OTHER PROTEINS

Free radicals formed during the reaction of H2O2 and metmyoglobin in t he presence of bovine serum albumin (BSA) were investigated using free ze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0-300 mu M) resulted in drastic changes in the characteristic fr eeze quench ESR signal of H2O2-activated metmyoglobin (perferryl prote in radical) under physiological conditions (pH = 7.4; I = 0.16). The r adical species formed during reaction of 100 mu M H2O2, 100 mu M metmy oglobin, and 200 mu M BSA have half-lives of approximately 13 min at 2 5 degrees C, in contrast to the perferryl protein radical that has a h alf-life of approximately 28 s at 25 degrees C. The radical species fo rmed in the presence of BSA were reactive towards ascorbate, glutathio ne, cysteine, and tyrosine. Substitution of BSA with defatted BSA, gam ma-globulin or beta-lactoglobulin also resulted in formation of long-l ived free radical species (half-lives: 13-18 min); however, the abilit y to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > gamma-globulin > beta-lacto globulin. The spin-trap alpha-phenyl-tert-butylnitrone (PEN) showed th e presence of transient protein radical species formed in the reaction between MMb, H2O2, and BSA. Transient radical species that could be p roposed as intermediates in the formation of the long-lived protein ra dicals detected by freeze-quench ESR. Dityrosine was formed in the rea ction between MMb, H2O2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction b etween H2O2-activated myoglobin and other proteins have major conseque nces on future interpretations of the significance of the perferryl pr otein radical in biological systems where proteins are abundant. (C) 1 997 Elsevier Science Inc.