Aging and some pathological conditions are associated with the accumulation of altered (inactive or less active) forms of enzymes. It was suggested that these agerelated alterations ref lect spontaneous changes in protein conformation and͞or posttranslational modifications (e.g., oxidation). Because changes in protein conformations are often associated with changes in surface hydrophobicity, we have examined the effects of aging and oxygen radicaldependent oxidation on the hydrophobicity of rat liver proteins. As a measure of hydrophobicity, the increase in f luorescence associated with the binding of 8-anilino-1-naphthalene-sulfonic acid to hydrophobic regions on the proteins was used. By this criterion, the hydrophobicity of liver proteins of 24-month-old rats was 15% greater than that of 2-month-old animals. Exposure of liver proteins to a metal-catalyzed oxidation system (ascorbate͞Fe(II)͞H 2 O 2 ) or a peroxyl radical generating system, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) led to increases of 2% or 30% in surface hydrophobicity, respectively. Treatment of liver proteins with the metal-catalyzed oxidation system led to a significant increase in reactive carbonyl content and to conversion of methionine residues to methionine sulfoxide residues. Treatment with AAPH led also to oxidation of methionine, tyrosine, and tryptophan residues and to the precipitation of some proteins. Dityrosine was detected in AAPH-treated protein, both the precipitate and supernatant fraction. The oxidation-dependent increase of hydrophobicity was correlated with an increase in the levels of methionine sulfoxide and dityrosine. These results suggest that oxidative modification of proteins may be responsible for the agerelated increase of protein surface hydrophobicity in vivo, and that the oxidation of methionine by an oxidative system may be an important event for the change of protein conformation.Covalent modification of proteins by oxidative systems have been implicated in various physiological and pathological conditions, such as aging, ischemia repurfusion and inflammatory disorders [for reviews, see Oliver et al. (1,2), and Stadtman (3, 4)]. These disorders are often associated with the accumulation of altered (inactive or less active) forms of various enzymes [for reviews, see Rothstein (5) and Oliver et al. (6)]. The age-related alterations may reflect spontaneous changes in protein conformations (7,8) or in protein oxidative modifications by reactive oxygen species (ROS) [for reviews, see Stadtman (9)]. The latter possibility is consistent with the facts that (i) many enzymes that accumulate as altered forms during aging are highly susceptible to modification by ROS; (ii) the ROS-mediated modifications of an enzyme can provoke changes in thermostability (10) and susceptibility to proteolytic degradation (10) similar to those that occur during aging (11); (iii) the level of oxidatively damaged protein, as measured by the presence of reactive carbonyl groups, increases with age (2). By whatever mechan...