SPECTRAL PROPERTIES OF ENVIRONMENTALLY SENSITIVE PROBES ASSOCIATED WITH HORSERADISH-PEROXIDASE

The environmentally sensitive fluorescent probes B-propionyl-2-(N,N-di methylamino)naphthalene (PRODAN) and N,N-dimethylamino)-6-naphthoyl-4- tmns-cyclohexanio acid (DANCA) form complexes with the heme binding si te of apohorseradish peroxidase. The dissociation constants of the PRO DAN 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 Fro m comparison of the steady state fluorescence spectra of PRODAN and DA NCA in solvents of varying dielectric constants, and in the apohorsera dish peroxidase complex, we conclude that the heme binding site of hor seradish peroxidase is relatively polar. The lifetimes of PRODAN and D ANCA in organic solvents of varying polarities can be fit to single ex ponential decays. However, the lifetimes of PRODAN acid DANCA associat ed with apohorseradish peroxidase, determined using a background subtr action method to correct for the non-negligible fluorescence of unboun d probe, fit best to a distribution of lifetime values. We attribute t hese 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. I n 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.