CONFORMATIONAL SUBSTATES OF APOPROTEIN OF HORSERADISH-PEROXIDASE IN AQUEOUS-SOLUTION - A FLUORESCENCE DYNAMICS STUDY

Detailed picosecond time-resolved fluorescence studies on the apoprotein of horseradish peroxidase (apoHRP) have been carried out by time-correlated single photon counting technique. The fluorescence decay was best described by a three-exponential model. Lifetime distributions recovered by maximum entropy method showed three bands which agreed with the discrete exponential model. Decay-associated spectra and acrylamide quenching studies revealed existence of multiple conformational substates of the protein resulting in multiexponential fluorescence decay of the single tryptophan in apoHRP. Time-resolved studies on apoHRP with an anionic porphyrin bound at its surface showed efficient energy transfer from the tryptophan to the porphyrin moiety corresponding to an energy transfer distance of similar to 15 Angstrom. Fluorescence anisotropy decay of apoHRP suggested a fast internal motion of the tryptophan residue along with a slower motion corresponding to global movement of the protein.