COMPARISON OF SPECTROPHOTOMETRIC AND AMPEROMETRIC RATE PARAMETERS OF ENZYMATIC-REACTIONS

In the context of current interest in electroanalytical sensors for biomedical applications, enzyme kinetic parameters obtained amperometrically were compared with those determined spectrophotometrically for ethanol oxidation catalyzed by alcohol dehydrogenase. The amperometry was conducted at a platinum rotated disk electrode, utilizing hexacyanoferrate(III/II) as an electron shuttle between the enzyme-catalyzed redox reaction and the electrode. Overall good agreement was seen between the results obtained by the two methods except for V(max). The amperometric Michaelis constant, K(M), for ethanol was 3.2 mM, comparable to a reported value of 3 under similar experimental conditions. The amperometric turnover rate, k(cat), was 448 s-1, while the spectrophotometric literature assignment is 450 s-1. The amperometric K(M) for cosubstrate NAD was 0.80 mM versus spectrophotometric literature values ranging from 0.1 to 1 mM. On the other hand, the amperometric maximum initial reaction velocity, V(max), was 15% lower than its spectrophotometric counterpart.