Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity

Mitochondrial complex 1 exhibits some peculiar and poorly understood featur es regarding the effects of detergents on activity and sensitivity to hydro phobic inhibitors that are not seen with other membrane complexes using ubi quinone as a substrate. Therefore, we investigated the interaction of compl ex I from bovine heart mitochondria with different types of detergents by m onitoring activity, degree of inhibition and inhibitor binding in the prese nce of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-et her detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex 1 and compete with classical complex I inhibitors for a common b inding domain. These findings were used to develop a novel large-scale chro matographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was p urified by selective solubilization in Triton X-100 and subsequent hydroxyl apatite, ion-exchange and gel-exclusion chromatography. By switching deterg ents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatogr aphy the procedure yields highly pure, monodisperse and fully inhibitor-sen sitive enzyme. (C) 2000 Elsevier Science B.V. All rights reserved.