PROBING PH AND PRESSURE EFFECTS ON THE APOMYOGLOBIN HEME POCKET WITH THE N-DIMETHYLAMINO)-6-NAPHTHOYL-4-TRANS-CYCLOHEXANOIC ACID FLUOROPHORE

The environmentally sensitive fluorophore N-dimethylamino)-6-naphthoyl -4-trans-cyclohexanoic acid (DANCA) has been used to probe the apomyog lobin heme pocket. The unexpected polarity of this domain is generally interpreted as arising from dynamic dipolar relaxation of the peptide dipoles surrounding the heme pocket, In the present work we reexamine the photophysical properties of DANCA in a variety of solvents and co mplexed with apomyoglobin (apoMb) to further probe the heme pocket env ironment as a function of external solvent conditions, Absorption and excitation spectra in a number of solvents are consistent with the wel l-known pi <-- pi (LE) and pi* <-- n (CT) electronic absorption trans itions observed for naphthylamine derivatives. Dual emission is also a well-documented property of such derivatives, Based on the time scale of the heterogeneity in the decay of the DANCA fluorophore observed i n a series of solvents, we propose that the emission properties of DAN CA in apoMb are not uniquely attributable to dynamic relaxation events , but also reflect dual emission from both a long-lived, red CT state and the shorter-lived, blue LE state. The pH studies in the range of p H 5-9 of the emission properties of DANCA in apoMb support this hypoth esis, They also suggest a specific interaction of DANCA with one or bo th of the pocket histidyl residues, which leads to a drastic static qu enching and red shift of the bound DANCA fluorescence upon protonation . Similar effects are observed with increasing pressure, indicating th at these two perturbations alter the DANCA-apoMb complex in a similar fashion. The pressure-induced form of the protein is distinct both ene rgetically and structurally from the previously characterized acid int ermediate, in that it is populated above pH 5 and retains a significan t degree of integrity of the heme pocket.