Je. Brunet et al., HYDRODYNAMICS OF HORSERADISH-PEROXIDASE REVEALED BY GLOBAL ANALYSIS OF MULTIPLE FLUORESCENCE PROBES, Biophysical journal, 66(2), 1994, pp. 446-453
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.