Steady-state and time-resolved fluorescence of Esperase®:: comparison with the X-ray structure in the region of the two tryptophans

Fluorescence emission properties of the alkaline protease Esperase(R) have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase(R) was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase(R) (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gin 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase(R) are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca2+-binding sites. (C) 1998 Elsevier Science B.V. All rights reserved.