The importance of life-time changes in fluorescence depolarization.

The rotational relaxation time, rho, calculated from measurements of fluorescence depolarization, is clearly dependent on the assumed mean life-time, tau, of the excited state. However, variations in tau with experimental conditions (temperature and solvent composition) occur and the effect of such alterations in tau is demonstrated. In particular it should be noted that, unless life-time changes can be excluded, the occurrence of linear plots of reciprocal degree of polarization against the temperature/viscosity ratio does not necessarily indicate the absence of intramolecular freedoms. An attempt to correct for such life-time changes by measurement of the fluorescence intensity is made for the bovine serum albumin-1-dimethyl-aminonaphthalene-5-sulphonyl chloride system. The value of rho/3tau thus obtained for this system at 20 degrees is approx. 4.7, as against approx. 3.4 obtained by several workers in the absence of life-time corrections.