Fluorescene Difference Decay Curves: Resolution Of Complex Decays

Publisher Website
Google Scholar
Abstract
The use of fluorescence difference decay curves was explored as a way to isolate the decays of components in a complex system. Time-correlated single photon methodology was used to subtract a reference curve from a sample curve to yield a difference decay curve. To test the feasibility of this method, a model 3-component system (6-carboxyfluorescein, pyranine and 1-dimethyl-aminonaphthalene-5-sulfonate) was examined. From this complex fluorescence decay curve, the individual decays were obtained by subtracting the appropriate binary solution decay curves. These differences curves coincided with those of the single component systems. Stringent requirements included: use of the same instrumental settings for all solutions, low counting efficiency, avoidance of inner filter effects, absence of energy transfer and lack of chemical interaction between components. The difference decay method was applied to sequential dansylation of serum albumin. Lifetimes of the first 2 dyes bound were longer than those of the 3rd. Dynamic quenching of quinine fluorescence by Cl- was also determined. When the reference differed from the sample only by having a shorter lifetime, the difference decay curve had a characteristic shape. Quenching of intrinsic protein fluorescence by acrylamide using bovine serum albumin and liver alcohol dehydrogenase was also examined. Of the two Trp in each protein, one was preferentially quenched and its decay curve was obtained by difference.