RESOLUTION OF MULTIPHASIC REACTIONS BY THE COMBINATION OF FLUORESCENCE TOTAL-INTENSITY AND ANISOTROPY STOPPED-FLOW KINETIC-EXPERIMENTS

Multiphasic kinetics are often observed in stopped-flow investigations . To characterize further these kinetic phases, we have developed a me thodology whereby fluorescence total intensity and anisotropy stopped- flow data can be combined in a single analysis. Fluorescence total int ensity and anisotropy are highly interrelated and contain two very com plementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whe reas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescen ce quantum yield increases, the observed rate of anisotropy change wil l be more rapid than the total-intensity change, whereas in cases in w hich the total intensity decreases, the observed change in anisotropy will lag behind. In ail cases, with quantum yield changes the stopped- flow anisotropy signals cannot be fit with models consisting of expone ntials. Case studies examining these effects are described for the pro tein folding/refolding transitions of Staphylococcal nuclease and phos phoglycerate kinase. A multiphasic DNA exonuclease reaction using bact eriophage T4 DNA polymerase is also examined. In all of these cases, c ombined analysis of both data types revealed insights into reaction me chanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transien tly populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reaction s in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.