Determination of the orientation distribution of adsorbed fluorophores using TIRF. II. Measurements on porphyrin and cytochrome c.

The theory for determination of the orientation of adsorbed fluorescent molecules using total internal reflection fluorescence, as explained in part I of this series, is illustrated by measurements on adsorbed tetramethylpyridinium porphyrin (H2TMPyP) and porphyrin cytochrome c molecules. The results are encouraging, although for porphyrin cytochrome c the scatter in the obtained order parameters is substantial. For H2TMPyP molecules adsorbed on glass the orientation distribution depends on the solution concentration. At low concentration, the H2TMPyP molecules are more or less randomly oriented, whereas at high concentrations a broad distribution around an angle of 46 degrees between the porphyrin plane and surface was found. For cytochrome c adsorbed on glass and indium tin oxide it was impossible to interpret the data in terms of orientation distributions because of the scatter in the results. The total fluorescence as a function of the polarization angle psi 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 distribution seems to be independent on the (imposed) electrical potential of the interface.