Intrinsic molecular fluorescence of lactate dehydrogenase: an analytical alternative for enzymic determination of pyruvate

A method for the enzymic determination of pyruvate based on changes in the fluorescence intensity of lactate dehydrogenase (LDH) is described. These changes are due to the differential quenching effect produced by NAD and NADH on the LDH fluorescence. The NADH quenching is due to both an inner filter effect and LDH-NADH complex formation; the LDH-NADH complex is also fluorescent. However, the NAD quenching is based only on the inner filter effect. From these suppositions, the equilibrium constant of the reaction and the formation constant of the LDH-NADH complex were obtained. Given this, an appropriate analytical signal for the quantification of pyruvate and a mathematical model explaining the effect of each parameter are proposed. The linear response range of the method depends on the NADH concentration used during the determination; it is possible to determine pyruvate concentrations down to 8.8 x 10(-7) mol dm-3. The method was applied to the determination of pyruvate in synthetic blood samples with good accuracy.