Optimizing enzymatic cycling assays: spectrophotometric determination of low levels of pyruvate and L-lactate.
A kinetic analysis of enzymatic cycling systems covering the whole course of the reaction, i.e., the transient phase and the steady state, is presented. The cost of enzymatic cycling assays is minimalized by using equations to calculate the smallest amount of enzymes which should be used to obtain a given rate constant of the cycle. The model chemical system chosen for illustration purposes involved the determination of pyruvate and/or L-lactate via the coupling of L-lactate dehydrogenase and L-lactate oxidase cycling and NADH mediation. The method is simple although thorough and can be applied to any technique that uses enzymatic cycling.
Valero
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E
Departamento de Química-Física, Universidad de Castilla-La Mancha, Albacete, E-02071, Spain.
García-Carmona
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F
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000032:analysis*; D019344:Lactic Acid; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008956:Models, Chemical; D009132:Muscles / Q000737:chemistry; D011773:Pyruvates / Q000032:analysis*; D019289:Pyruvic Acid; D011817:Rabbits; D012680:Sensitivity and Specificity; D013053:Spectrophotometry
D007773:Lactates; D011773:Pyruvates; D019344:Lactic Acid; D019289:Pyruvic Acid; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase