2,4-Dinitrophenol: Lack of Interaction with High-Energy Intermediates of Oxidative Phosphorylation

If the uncoupling agent 2,4-dinitrophenol is added just prior to addition of the phosphate acceptor adenosine diphosphate to rat liver mitochondria previously incubated with substrate, orthophosphate, and oxygen, steady-state phosphorylation is abolished, whereas the initial phase of rapid phosphorylation, the so-called "adenosine triphosphate jump," is undisturbed. This leads to the conclusion that uncoupling agents operate by interfering with the synthesis of high-energy intermediates, not by hydrolyzing or otherwise inactivating them.