Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

Abstract

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed.

Growth and Physiological Responses of Adenophora triphylla (Thunb.) A.DC. Plug Seedlings to Day and Night Temperature Regimes

“…Clearly, an in-depth study of the expression and activity of the various antioxidant isozymes under temperature regimes is necessary to figure out antioxidant system and self-defending mechanism in plant. Native-polyacrylamide gel electrophoresis (native-PAGE) is an effective technology used to separate isozymes while retaining aspects of their activity [35,36]. And the band intensity was closely dependent on the activities of isozymes.…”