INCREASED RESOLUTION AMONG PHOSPHORESCENCE DECAY COMPONENTS OF LADH BY THE USE OF QUENCHING

摘要

The room temperature phosphorescence decay of Horse Liver Alcohol Dehydrogenase (LADH) was analyzed with continuous lifetime distribution models such as the Exponential Series and Maximum Entropy Methods, revealing the existence of a broad distribution of phosphorescence lifetimes. Possibly reflecting the existence of two or more conformational species that do not rapidly interconvert on a time scale shorter than seconds. In order to gain insight into the underlying reason for the lifetime distribution, we performed a series of quenching experiments on LADH phosphorescence. When quenching data is presented in terms of a distribution of decay rate constants (rather than lifetimes) it is easy to show that quenching of the phosphorescence by mechanisms that do not distinguish between protein species will result in a uniform increase in the decay rate constant without affecting the width of the distribution. An example would be a Forster quenching mechanism if the components within the distribution have identical overlap integrals with the energy transfer partner. Conversely, if the species responsible for the distribution have a differential susceptibility to the quencher, an increase in the mean rate constant and a change in the distribution width will occur. Thus, a quencher that diffuses differentially into various protein conformers is expected to cause a change in the width of the phosphorescence distribution. This change in width provides information on the relative efficiency of quenching of conformers. Using a number of quenchers, one may resolve components within the distribution of conformational states by analyzing the dependence of the width of the phosphorescence lifetime distribution on quencher concentration.