Lactate dehydrogenase kinetics and inhibition using a microplate reader

Abstract

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(I) . 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.

Bringing the excitement and motivation of research to students; Using inquiry and research‐based learning in a year‐long biochemistry laboratory

“…Students receive a basic protocol and are asked to design their own experiments to determine Km and Vmax for the assay. For those laboratories that meet more than once a week, a much deeper kinetic analysis can be conducted using an inquiry format [20, 21]. Week 14 is typically spent working with groups as they design their procedure, while week 15 is devoted to conducting the experiment and graphing the results.…”

New ideas for an old enzyme: A short, question‐based laboratory project for the purification and identification of an unknown LDH isozyme

“…The purification and analysis project we have described here could easily be done as a module of a larger project. In particular, LDH is commonly used to study enzyme kinetics, and several good sets of experiments have been published on this [6, 7]. We have found this project to be enjoyable for the students to perform and for the faculty to teach.…”

“…Second, each purification technique works by exploiting a different biochemical property of the enzyme so that a number of fundamental biochemical principles are demonstrated by the experiments. Lactate dehydrogenase (LDH) has long been a popular enzyme to work with in student laboratories [6, 7]. The starting material for its purification from mammalian tissue (bovine or porcine skeletal muscle, heart, and liver) is easy to obtain, and the enzyme is relatively stable in student hands.…”

Purification and Characterisation of Lactate Dehydrogenase: An Undergraduate Biochemistry Laboratory Experiment

“…Hydrazine favours a second fast, non enzymatic reaction that converts pyruvate to pyruvate-hydrazone. Pyruvate-hydrazone cannot bind as well as pyruvate to the active site of LDH, thus, the coupled reaction prevents reversibility and also decreases the likelihood of product inhibition [10]. This is clearly seen in Figure 8 for concentrations over 20 mM L-lactate.…”