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

This paper presents a procedure for fluorometric-enzymatic lactate det ermination based on the modification of the fluorometric properties of the enzyme L-lactic dehydrogenase (cytochrome b2), during the enzymat ic oxidation of the analyte with ferricyanide. During the reaction one can observe an irreversible fall in the intensity of the enzyme's flu orescence, the rate of which is proportional to the concentration of t he lactate. The source of this signal has been investigated and it has been shown that, besides the formation of a complex between the enzym e and the ferricyanide (the constant of which can be determined), this signal loss can be explained by simultaneous inner filter effects cau sed by the ferricyanide and the ferrocyanide (generated in the enzymat ic reaction). A mathematical model has been developed which makes it p ossible to establish a linear response between the enzyme's analytical signal of fluorescence and the concentrations of the lactate, the cyt ochrome, and the ferricyanide. The procedure makes it possible to dete rmine the lactate in concentrations ranging from 0.2 to 45 mg/L. Deter mination of the analyte has been carried out in milk samples with grea t precision and accuracy.