DETERMINATION OF THE ORIENTATION DISTRIBUTION OF ADSORBED FLUOROPHORES USING TIRF .2. MEASUREMENTS ON PORPHYRIN AND CYTOCHROME-C

The theory for determination of the orientation of adsorbed fluorescen t molecules using total internal reflection fluorescence, as explained in part I of this series, is illustrated by measurements on adsorbed tetramethylpyridinium porphyrin (H(2)TMPyP) and porphyrin cytochrome c molecules. The results are encouraging, although for porphyrin cytoch rome c the scatter in the obtained order parameters is substantial. Fo r H(2)TMPyP molecules adsorbed on glass the orientation distribution d epends on the solution concentration. At low concentration, the H(2)TM PyP molecules are more or less randomly oriented, whereas at high conc entrations a broad distribution around an angle of 46 degrees between the porphyrin plane and surface was found. For cytochrome c adsorbed o n glass and indium tin oxide it was impossible to interpret the data i n terms of orientation distributions because of the scatter in the res ults. The total fluorescence as a function of the polarization angle P si of the incident light beam corresponds to an average angle between the porphyrin group and the surface of 30 degrees-40 degrees. Despite the strong electric dipole moment of the protein, the orientation dist ribution seems to be independent on the (imposed) electrical potential of the interface.