Lactate dehydrogenase kinetics and inhibition using a microplate reader.

Area of Science:

• Biochemistry
• Enzyme Kinetics
• Biotechnology

Background:

• Lactate dehydrogenase (LDH) is crucial in cellular metabolism.
• Traditional enzyme kinetics assays can be time-consuming.
• Microplate spectrophotometry offers rapid, high-throughput data acquisition.

Purpose of the Study:

• To develop and validate a microplate-based laboratory experiment for enzyme kinetics.
• To investigate the inhibition of lactate dehydrogenase (LDH) using novel methods.
• To provide students with hands-on experience in enzyme kinetics and data analysis.

Main Methods:

• Utilized a microplate spectrophotometer for rapid absorbance readings.
• Prepared 12 samples at a time for efficient data collection.
• Collected kinetic data at five substrate concentrations with and without two inhibitor concentrations.
• Employed urea as a novel quench reagent.

Main Results:

• Students successfully generated Michaelis-Menten and Lineweaver-Burk plots.
• Calculated key kinetic parameters including Vmax, apparent Vmax, Km, apparent Km, kcat, and Ki.
• Demonstrated competitive inhibition by oxalic acid and noncompetitive inhibition by oxamic acid.

Conclusions:

• The microplate format provides an efficient and effective method for teaching enzyme kinetics.
• The assay is relevant for students interested in medical careers and metabolism.
• Novel features include the microplate format and the use of urea as a quench reagent.