Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

The environmentally sensitive fluorescent probes 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) and 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanioc acid (DANCA) form complexes with the heme binding site of apohorseradish peroxidase. The dissociation constants of the PRODAN and DANCA complexes were determined from anisotropy titration data to be approximately 8.7 x 10(-5) and 3.3 x 10(-4) M, respectively. From comparison of the steady state fluorescence spectra of PRODAN and DANCA in solvents of varying dielectric constants, and in the apohorseradish peroxidase complex, we conclude that the heme binding site of horseradish peroxidase is relatively polar. The lifetimes of PRODAN and DANCA in organic solvents of varying polarities can be fit to single exponential decays. However, the lifetimes of PRODAN and DANCA associated with apohorseradish peroxidase, determined using a background subtraction method to correct for the non-negligible fluorescence of unbound probe, fit best to a distribution of lifetime values. We attribute these lifetime distributions to microenvironmental heterogeneity which is also consistent with the observed dependence of the emission maxima of PRODAN-apohorseradish peroxidase upon the excitation wavelength. In neither the PRODAN nor the DANCA case was evidence found in the time-resolved data for relaxation of the protein matrix around the excited state probe dipole.

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