Free radical damage to proteins: The influence of the relative localization of radical generation, antioxidants, and target proteins

Abstract

Free radicals were generated at known rates in the aqueous phase (by means of 2,2′-azobis (2-amidinopropane) dihydrochloride [AAPH]) and in a membranous (lipid) phase (by means of 2,2′-azobis (2,4-dimethylvaleronitrile [AMVN]). A soluble protein (bovine serum albumin; BSA), and membranes of lysed mitochondria containing radioactively labeled monoamine oxidase (MAO), were exposed to the resultant radical fluxes. Antioxidants were added to the system, either in the aqueous phase (Trolox) or in a liposomal membrane phase (α-tocopherol). Protein damage was assessed as tryptophan oxidation and conformational changes in tryptophan fluorescence of the soluble protein. BSA, and as framentation of both BSA and monoamine oxidase. Radicals generated in the acquous phase, by AAPH, were affective in damaging BSA and MAO. Radicals generated within the liposome membrane phase (by AMVN) were less effective against BSA than those deriving from AAPH. Liposomal AMVN radicals could damage MAO, present in a separate membranous phase, though again, less effectively than could APPH-derived radicals. BSA could be protected by Trolox, the aqeous soluble antioxidant, but hardly by tocopherol itself. Damage to MAO was limited by Trolox, and also by the hydrophobic antioxidant, tocopherol. Damaging reactions due to radicals generated in a membrane phase were significantly accelerated when the membrane was peroxidizable (soybean phosphatidylcholine) rather than nonperoxidizable (saturated dimyristoyl phosphatidylcholine). Thus lipid radicals also played some role in protein damage in these systems. BSA was attacked similarly in the presence of liposomes by AAPH. Correspondingly, BSA could inhibit the peroxidation of liposomes induced by AAPH and less efficiently that induced by AMVN. It is concluded that the relative localization of radical generation, antioxidants, and protein targets has a major influence on the extent of radical attact on proteins and membranes.