Assays of d-Amino Acid Oxidases
d-Amino acid oxidase and d-aspartate oxidase are two well-known FAD-containing flavooxidases that catalyze the same reaction (the oxidative deamination) on different d-amino acids. d-aspartate oxidase is specific for acidic d-amino acids (i.e., d-aspartate and d-glutamate) and d-amino acid oxidase is active on neutral and polar d-amino acids (a low activity is also detected on basic d-amino acids). The assay of these flavoenzymes is of utmost importance in different fields because d-amino acids are common constituents of bacterial cell walls, are present in foods and because free d-serine and d-aspartic acid were identified in brain and peripheral tissues of mammals. In this chapter, we report on the most used methods employed to assay the activity of d-amino acid oxidase and d-aspartate oxidase. Interestingly, their activity can be followed using different assays, namely d-amino acid or oxygen consumption, α-keto acid or ammonia production, or using artificial dyes as final indicator of the flavin redox reaction.
Key wordsEnzymatic assay d-Amino acid oxidase d-Aspartate oxidase Analytical detection
The works carried out in the laboratory of L. Pollegioni were supported by grants from Fondo di Ateneo per la Ricerca (University of Insubria) and from Fondazione Cariplo. LP thanks the support of Consorzio Interuniversitario per le Biotecnologie (CIB) and of Centro di Ricerca in Biotecnologie per la Salute Umana (University of Insubria). The work carried out in the laboratory of G. Tedeschi and A. Negri was supported by grants from Ministero dell’Università e della Ricerca Scientifica and from the Consiglio Nazionale delle Ricerche (Italy).
- 13.Fonda M L, Anderson B M (1969) D-amino acid oxidase. IV. Inactivation by maleimides. J Biol Chem 244, 666–674.Google Scholar
- 15.Negri A, Tedeschi G, Ceciliani F, Ronchi S (1999) Purification of beef kidney D-aspartate oxidase overexpressed in E. coli and characterization of its redox potentials and oxidative activity towards agonists of excitatory amino acid receptors. Biochim Biophys Acta 143, 212–222.CrossRefGoogle Scholar
- 18.Tedeschi G, Negri A, Bernardini G et al. (1999) D-Aspartate oxidase is present in ovaries, eggs and embryos but not in testis of Xenopus laevis. Comp Biochem Physiol 124B, 489–494.Google Scholar