Kinetics of Papain: An Introductory Biochemistry Laboratory Experiment

Enzyme kinetics experiments are popular in the undergraduate laboratory. These experiments have pedagogic value because they reinforce the concepts of Michaelis-Menten kinetics covered in the lecture portion of the course and give students the experience of calculating kinetic constants from data they themselves have generated. In this experiment, we investigate the kinetics of the thiol protease papain. The source of the papain is commercially available papaya latex. A specific substrate, Nσ-benzoyl-arginine-p-nitroanilide (BAPNA), is used, which takes advantage of the fact that papain interacts with a phenylalanine residue two amino acids away from the peptide bond cleaved. Upon hydrolysis by papain, a bright yellow product is released, p-nitroaniline. This allows the reaction to be monitored spectrophotometrically by measuring the rate of formation of the p-nitroaniline product as a function of the increase in absorbance of the solution at the σmax of p-nitroaniline (400 nm) over time at various substrate concentrations. These data are used to plot a Lineweaver-Burk plot from which the vmax and KM are obtained. If time permits, students carry out additional investigations in which ε of p-nitroaniline is measured, the enzyme solution protein concentration is measured, the enzyme purity is evaluated by SDS-PAGE, and a pH-rate profile is constructed from experimental data.