Buffer effects on EcoRV kinetics as measured by fluorescent staining and digital imaging of plasmid cleavage

We have developed a protocol to quantify polymer DNA cleavage which replace s the traditional radiolabeling and scintillation counting with fluorescent staining and digital imaging. This procedure offers high sensitivity, spee d, and convenience, while avoiding waste and error associated with traditio nal P-32 radiolabeling, This protocol was used to measure cleavage of pBR32 2 plasmid DNA by EcoRV, a type II restriction enzyme. EcoRV was found to ex hibit an order of magnitude difference in binding in two apparently similar buffers used in previous investigations. To determine the origin of this e ffect, we measured reaction kinetics in buffers of different chemical natur e and concentration: Tris, bis-Tris propane, Tes, Hepes, and cacodylate, We found that buffer concentration and identity had significant effects on Ec oRV reaction velocity through large changes in specific binding and nonspec ific binding (reflected in the Michaelis constant K-m and the dissociation constant for nonspecific binding K-ns). There were only small changes in V- max. The source of the buffer effect is the protonated amines common to man y pH buffers. These buffer cations likely act as counterions screening DNA phosphates, where both the protonated buffer structure and concentration af fect enzyme binding strength. It appears that by choosing anionic buffers o r zwitterionic buffers with a buried positive charge, buffer influence on t he protein binding to DNA can be largely eliminated. (C) 1999 Academic Pres s.