RAMAN-STUDY OF ENZYME-REACTIONS USING POTASSIUM FERRICYANIDE AS A REACTION MEDIATOR - QUANTITATIVE-ANALYSIS OF SUBSTRATES AND MEASUREMENT OF ENZYME-ACTIVITY FOR GLUCOSE-OXIDASE AND LACTATE OXIDASE

This paper describes a Raman study of enzyme reactions that use potass ium ferricyanide as a reaction mediator, The enzyme reaction systems i nvestigated consisted of glucose oxidase (GOD), glucose, and potassium ferricyanide, and of lactate oxidase (LOD), lactate, and potassium fe rricyanide. Raman spectra were measured for the above enzyme systems e very 2 s to monitor the progress of the enzyme reactions at real time. The observed Raman spectra showed only three peaks at 2186, 2095, and 2022 cm(-1) due to C=N stretching modes of potassium ferricyanide (21 86 cm(-1)) and potassium ferrocyanide (2095 and 2022 cm(-1)); with the progress of the enzyme reactions, the band at 2186 cm(-1) decreases w hile those at 2095 and 2022 cm(-1) increase. From the differentiation of the Raman intensity at 2022 cm(-1), we could calculate the velocity (V') of the intensity change that directly reflects the rate (V) of t he enzyme reactions. By plotting the velocity thus obtained (V') vs, t he concentration of the enzyme substrate ([S]), we were able to develo p a calibration curve to predict the concentration of the enzyme subst rate. For GOD, the correlation coefficient (R) and the detection limit of this calibration curve were 0.99 and 20 mg/dL, respectively. This detection limit was better than that obtained from conventional glucos e sensors. For LOD, we were able to determine Michaelis constant (K-m) from the maximum velocity (V-max). The method proposed here is applic able to various kinds of enzymes that use potassium ferricyanide as a reaction mediator.