The temperature dependence of fluorescence anisotropy, lifetime and differential tangent of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its polar trimethylammonium derivative (TMA-DPH) were investigated in cytoplasmic membranes of Bacillus subtilis. The fluorescence parameters were compared in the two types of membranes prepared from bacteria cultivated at 20 and 40 degrees C. Steady-state anisotropy measurements showed that within a broad range of temperatures, membranes cultivated at 20 degrees C exhibit significantly lower values than those prepared from cells cultivated at 40 degrees C. The temperature dependence of lifetime and differential tangent measurements (differential polarized phase fluorimetry) were fully consistent with steady-state anisotropy data of both DPH and TMA-DPH. The low anisotropy values in the case of TMA-DPH could be explained by a shorter lifetime and higher temperature-induced decrease as compared with DPH. Surprisingly, the temperature dependence of rotational rate R calculated according to the model of hindered rotations (Lakowicz 1983) gave misleading results. When increasing the temperature from 5 to 25 degrees C, a marked drop of rotational relaxation rate was observed. The minimum R values were measured between 25 and 30 degrees C and further increase of temperature (up to 60 degrees C) was reflected as increase of the R values. Therefore, a new model of "heterogeneous rotations" was developed. This model assumes that even at low temperatures (approaching 0 degrees C) where the differential tangent reaches zero, a fraction of fast rotating molecules exists. The ratio between fast and slowly rotating molecules may be expressed by this model, the newly calculated rotational rates are fully consistent with anisotropy, lifetime and differential tangent measurements and represent the monotonically increasing function of temperature.

Department of Microbiology Faculty of Science Charles University Prague

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