DYNAMIC FLUORESCENCE OF TRYPTOPHANYL RESIDUES IN LOW MOLECULAR WEIGHT MODEL COMPOUNDS AND PROTEINS

The tryptophanyl fluorescence decay of tuna apomyoglobin, a protein containing a single indole residue, has been compared to that of the monomeric tryptophanyl residue (NATA) in order to discriminate the effect determined by the protein matrix from those induced by physical and chemical agents. The fluorescence decay, obtained in the frequency domain, has been analyzed in terms of lifetime distributions having a Lorentzian shape. The results indicate that the incorporation of the chromophore into the protein matrix determines a broadening of the distribution pattern.
A further increase of the distribution width has been observed on guanidine-induced unfolding of the protein, whereas no effect has been detected for NATA in the presence of guanidine at concentrations as high as 6.0 M. These observations lead to the conclusion that the tryptophanyl fluorescence lifetime is influenced by the protein matrix even in the fully unfolded state.
The increase of temperature from 20 to 45_oC produces an enlargement of the distribution width of NATA and unfolded tuna apomyoglobin. In the same thermal range, the native protein exhibits a narrowing of the tryptophanyl lifetime distribution. This different behaviour has been explained in terms of a larger distribution of microenvironmental states, generated by the chromophore-solvent interaction (which is very limited in the native protein), at higher temperature.
This conclusion has been further corroborated by the observation that a temperature increase changes the weight of the various components which contribute to the total emission of the free amino acid tryptophan.