ELECTROCHEMICAL BIOASSAY USING APOENZYME- FLAVIN-ADENINE DINUCLEOTIDEINTERACTION FOR THE DETECTION OF FLAVIN-ADENINE DINUCLEOTIDE

We employed the commercially available D-amino acid oxidase (MW = 3800 0 g/mol) which requires one flavin-adenine dinucleotide (FAD) as a cof actor of activation. It can enzymatically catalyze the oxidation of D- amino acid to 2-oxoacid in the presence of H2O and O-2. In addition, t his enzyme produces NH3 and H2O2 during enzymatic reactions. This cofa ctor is essential for the activation of the apoenzvme. and the active enzyme produces electroactive H2O2 and NH3 when the cofactor is attach ed to the specific site of the apoenzvme. Therefore we prepared approp riate amounts of apoenzyme and DL-alpha-alanine in buffer solution (pH 8.30). H2O2 was produced by the enzymatic process in response to the addition of FAD. Then the concentration of FAD was estimated from the electrochemical signal of H2O2 at +600 mV vs. Ag/AgCl. This configurat ion discriminated between different concentrations of analyte in the r ange 0.84-4.2 mu M.