Lactate dehydrogenase kinetics and inhibition using a microplate reader

A lactate dehydrogenase (LDH) enzyme kinetics laboratory experiment has been developed in which students obtain kinetic data using a microplate spectrophotometer (reader). These instruments have the capability of reading absorbances of many samples in a very short time frame. In this experiment 12 samples are prepared at a time and the absorbances read in less than 1 min. In a 3-hr laboratory period, students collect data at five different substrate concentrations without inhibitor and also in the presence of two different concentrations of inhibitor. Students have enough time to repeat each part if they obtain too much scatter in their data. The enzyme examined, LDH, correlates with the study of metabolism and has particular relevance for students who are interested in medical careers. The LDH assay itself is not new, but the microplate format and the use of urea as a quench reagent are novel features. Students plot Michaelis-Menten and Lineweaver-Burk plots and calculate values for V_max, apparent V_max (V_max^app). K_m, apparent K_m (K_m^app), k _cat, and K_l. Students typically obtain results correctly showing that oxalic acid is a competitive inhibitor and oxamic acid is a noncompetitive inhibitor when lactate is the substrate of the reaction.