[23] Analysis of transmembrane redox reactions: Interaction of intra- and extracellular ascorbate species

The chapter discusses techniques that can be used to study ascorbate-related redox reactions across cell membranes. First, the quantification of intra- and extracellular ascorbate species is discussed. Subsequently, the chapter presents methods to establish the proper intra- or extracellular concentrations of some ascorbate species, as well as methods for the detection of redox reactions between these intra- and extracellular molecules. Ascorbate is stable as a solid, and should only be dissolved on the day of use, because of its susceptibility to oxidative degradation. Ascorbate oxidase (EC 1.10.3.3) is a useful enzyme in the study of ascorbate and its free radical. The enzyme can be purchased as a lyophilized solid. Ascorbate has a strong absorption band at 265 nm, allowing simple and convenient spectrophotometric quantification. Several different approaches are possible for the analysis of dehydroascorbic acid (DHA). Four of them are described in the chapter—namely, reduction of DHA, derivatization of DHA, radioactive labeling of DHA, and NMR and 13C-labeled DHA. It is mentioned that ESR spectroscopy is the most powerful technique for studying free radical molecules, such as ascorbate-free radical (AFR), and their interactions with the living cell.