Crowding effects on EcoRV kinetics and binding

The cytosol of the cell contains high concentrations of small and large mac romolecules, but experimental data are often obtained in dilute solutions t hat do not reflect in vivo conditions. We have studied the crowding effect that large macromolecules have on EcoRV cleavage by adding high-molecular-w eight Ficoll 70 to reaction solutions. Results indicate that Ficoll has sur prisingly little effect on overall EcoRV reaction velocity because of offse tting increases in V-max and K-m, and stronger nonspecific binding. The cha nges in measured parameters can largely be attributed to the excluded volum e effects on reactant activities and the slowing of protein diffusion. Covo lume reduction upon binding appears to reinforce nonspecific binding streng th, and k(cat) appears to be slowed by stronger nonspecific binding, which slows product release. The data also suggest that effective Ficoll particle volume decreases as its concentration increases above a few weight percent , which may be due to Ficoll interpenetration or compression.