Brain mitochondrial complex I inactivation by oxidative modification

In vitro oxidation of the brain mitochondrial complex I by the hydroxyl radical generating system ascorbate/Fe(III)/O-2 has been carried out. Complex I inactivation, by oxidation, has been studied using a method based on the resolution of proteins by blue native polyacrylamide gel electrophoresis (BN-PAGE), followed by total protein quantification by staining with Coomassie brilliant blue, in-gel activity quantification, and quantification of oxidized proteins by labelling with DIG-hydrazide and immunodetection with an anti-DIG-AP. Quantification was carried out by densitometry procedure. Our results show that oxidation is a continuous process, increasing rapidly at the beginning, reaching a plateau after 8 h of incubation. There is practically no inactivation until a threshold value of damage is reached. Below this, the complex activity is resistant to the aggression of oxygen-reactive substances and free radicals, but once the threshold value is passed, activity is lost rapidly. (C) 2000 Academic Press.