HYDRODYNAMICS OF HORSERADISH-PEROXIDASE REVEALED BY GLOBAL ANALYSIS OF MULTIPLE FLUORESCENCE PROBES

Previous fluorescence studies of horseradish peroxidase conjugated wit h protoporphyrin IX suggested that the protein behaved hydrodynamicall y as a prolate ellipsoid of axial ratio 3 to 1. The present study, des igned to further investigate the hydrodynamics of this protein, exploi ts a series of probes, noncovalently bound to the heme binding site of apohorseradish peroxidase, having different orientations of the excit ation and emission transition dipoles with respect to the protein's ro tational 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 sulfonat e). Time-resolved data were obtained using multifrequency phase fluoro metry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets w hile maintaining a constant molecular shape for the protein. The resul ts indicated that, in such analyses, probes exhibiting a single expone ntial decay and limited local motion have the major weight in the eval uation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to sign ificant uncertainties in such global treatments. By explicitly account ing for the effect of such local motion, however, the shape of the pro tein can be reliably recovered.