Buffer Effects onEcoRV Kinetics as Measured by Fluorescent Staining and Digital Imaging of Plasmid Cleavage

Abstract

We have developed a protocol to quantify polymer DNA cleavage which replaces the traditional radiolabeling and scintillation counting with fluorescent staining and digital imaging. This procedure offers high sensitivity, speed, and convenience, while avoiding waste and error associated with traditional32P radiolabeling. This protocol was used to measure cleavage of pBR322 plasmid DNA byEcoRV, a type II restriction enzyme.EcoRV was found to exhibit an order of magnitude difference in binding in two apparently similar buffers used in previous investigations. To determine the origin of this effect, we measured reaction kinetics in buffers of different chemical nature and concentration: Tris, bis-Tris propane, Tes, Hepes, and cacodylate. We found that buffer concentration and identity had significant effects onEcoRV reaction velocity through large changes in specific binding and nonspecific binding (reflected in the Michaelis constantKmand the dissociation constant for nonspecific bindingKns). There were only small changes inVmax. The source of the buffer effect is the protonated amines common to many pH buffers. These buffer cations likely act as counterions screening DNA phosphates, where both the protonated buffer structure and concentration affect enzyme binding strength. It appears that by choosing anionic buffers or zwitterionic buffers with a buried positive charge, buffer influence on the protein binding to DNA can be largely eliminated.