Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry

Abstract

The stationary fluorescence polarization and the differential phase delay of the polarized components of the fluorescence of 1-phenylnaphthylamine in Escherichia coli suspensions were measured before and after addition of colicin E1. Both sets of measurements register an increase in the rotational relaxation time of the fluorescent probe when colicin is present. These increases are absent in an E. coli mutant tolerant to colicin E1. The physical interpretation of the changes demands separate estimation of the fraction f2 of the emitting fluorophores that change their properties upon colicin addition and of the rotational relaxation time .rho.2 of this fraction, following the colicin-induced changes. By themselves, the steady state polarization observations permit only the conclusion that f2 must be in the range of 1-0.06 and the change in .rho.2/.tau. between 1.5 and a value larger than 10. Combination of the data of stationary polarization with those of differential phase fluorometry results in an important reduction in the uncertainty: f2 must be in the range 1-0.33 and the change in .rho.2/.tau. in the range 1.5-2.5.