Lactate Dehydrogenase Kinetics and Inhibition using a Microplate Reader


Chemistry and Biochemistry

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A lactate dehydrogenase (LDH) enzyme kinetics lab. expt. 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 expt. 12 samples are prepd. at a time and the absorbances read in less than 1 min. In a 3-h lab. period, students collect data at five different substrate concns. without inhibitor and also in the presence of two different concns. of inhibitor. Students have enough time to repeat each part if they obtain too much scatter in their data. The enzyme examd., LDH, correlates with the study of metab. 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 calc. values for Vmax, apparent Vmax (Vappmax), Km, apparent Km (Kappm), kcat, and kl. 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.