A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Abstract

The emerging importance of Peptidyl-tRNA hydrolase (Pth) enzymes necessitates the need for a widely applicable functional assay to further studies of this important enzyme family. Previously reported methods for monitoring Pth function suffer from limitations of cost, time, substrate availability, and application compatibility. Herein we present a new method for the rapid and precise characterization of Pth activity. The method is applicable for use with specific or bulk peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives. The method also allows for semi-automated quantitative assessment of peptidyl-tRNA cleavage. No specialized equipment, harmful reagents, or time-consuming techniques are required. We use the new method to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.