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Biophysical Journal 70: 1996-2000 (1996)
© 1996 the Biophysical Society

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Improved differentiation between luminescence decay components by use of time-resolved optical activity measurements and selective lifetime modulation.

J A Schauerte, A Gafni and D G Steel

Department of Biological Chemistry, University of Michigan, Ann Arbor 48109, USA.

ABSTRACT

The analysis of luminescence decay experiments from proteins is typically modeled as a combination of independent first-order decay functions. However, Poisson noise in the photon counting experiment limits the ability of this approach to resolve decay components from separate lumiphores with similar lifetimes. To provide further differentiation, we incorporate time-resolved circular polarization of luminescence, an additional independent observable, into the analysis. In the simplest case, for example, each lumiphore's chirality is assumed to be time independent and is determined by the position of the lumiphore with respect to the surrounding chiral environment within the protein. In this paper, we describe the analysis of simultaneously recorded time-resolved luminescence and circularly polarized luminescence data to obtain improved temporal resolution. When combined with selective dynamic luminescence quenching, in a model system comprising a mixture of Tb/transferrin and Tb/conalbumin, we demonstrate resolution between two decay components with a lifetime difference of 7% and a difference in emission anisotropy of 5 X 10(-2). Evidence for the improved discrimination is further demonstrated by the increase in curvature of the chi 2 surface that results from the additional information.






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Copyright © 1996 by the Biophysical Society.