Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.
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.
Brunet
J E
JE
Instituto de Química, Universidad Católica de Valparaiso, Chile.
Vargas
V
V
Gratton
E
E
Jameson
D M
DM
D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D008956:Models, Chemical; D011487:Protein Conformation; D011524:Protoporphyrins / Q000737:chemistry
D005456:Fluorescent Dyes; D011524:Protoporphyrins; D006418:Heme; C028025:protoporphyrin IX; D006735:Horseradish Peroxidase