FLUOROMETRIC ENZYMATIC LACTATE DETERMINATION BASED ON ENZYME CYTOCHROME-B(2) FLUORESCENCE

This paper presents a procedure for fluorometric-enzymatic lactate determination based on the modification of the fluorometric properties of the enzyme L-lactic dehydrogenase (cytochrome b2), during the enzymatic oxidation of the analyte with ferricyanide. During the reaction one can observe an irreversible fall in the intensity of the enzyme's fluorescence, the rate of which is proportional to the concentration of the lactate. The source of this signal has been investigated and it has been shown that, besides the formation of a complex between the enzyme and the ferricyanide (the constant of which can be determined), this signal loss can be explained by simultaneous inner filter effects caused by the ferricyanide and the ferrocyanide (generated in the enzymatic reaction). A mathematical model has been developed which makes it possible to establish a linear response between the enzyme's analytical signal of fluorescence and the concentrations of the lactate, the cytochrome, and the ferricyanide. The procedure makes it possible to determine the lactate in concentrations ranging from 0.2 to 45 mg/L. Determination of the analyte has been carried out in milk samples with great precision and accuracy.