INTRINSIC MOLECULAR FLUORESCENCE OF LACTATE-DEHYDROGENASE - AN ANALYTICAL ALTERNATIVE FOR ENZYMATIC DETERMINATION OF PYRUVATE

A method for the enzymic determination of pyruvate based on changes in the fluorescence intensity of lactate dehydrogenase (LDH) is describe d. 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-N ADH complex is also fluorescent, However, the NAD quenching is based o nly on the inner filter effect. From these suppositions, the equilibri um constant of the reaction and the formation constant of the LDH-NADH complex were obtained, Given this, an appropiate analytical signal fo r the quantification of pyruvate and a mathematical model explaining t he effect of each parameter are proposed, The linear response range of the method depends on the NADH concentration used during the determin ation; 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 p yruvate in synthetic blood samples with good accuracy.