Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin

Fluorescence quenching and time-resolved fluorescence studies have been per formed on the galactose-specific lectin purified from snake gourd (Trichosa nthes anguina) seeds, in order to investigate the tryptophan accessibility and environment in the native protein and in the presence of bound ligand. Estimation of the tryptophan content by N-bromosuccinimide modification in the presence of 8 M urea yields four residues per dimeric molecule. The emi ssion spectrum of native lectin in the absence as well as in the presence o f 50 mM methyl-alpha-D-galactopyranoside (Me alpha Gal) shows a maximum aro und 331 MI, which shifts to 361.8 nm upon reduction of the disulfide bonds and denaturation with 8 M urea, indicating that all four tryptophan residue s in the native state of this protein are in a hydrophobic environment. The extent of quenching that is observed is highest with acrylamide, intermedi ate with succinimide, and low with Cs+ and I-, further supporting the idea that the tryptophan residues are predominantly buried in the hydrophobic co re of the protein. The presence of Me alpha Gal (50 mM) affects the quenchi ng only marginally. Time-resolved fluorescence measurements yield bi-expone ntial decay curves with Lifetimes of 1.45 and 4.99 ns in the absence of sug ar, and 1.36 and 4.8 ns in its presence. These results suggest that the try ptophan residues are not directly involved in the saccharide binding activi ty of the T, anguina lectin. Of the four quenchers employed in this study, the cationic quencher, Cs+, is found to be a very sensitive probe for the t ryptophan environment of this lectin and may be useful in investigating the environment of partially buried tryptophan residues and unfolding processe s in other proteins as well. (C) 1999 Elsevier Science S.A. AU rights reser ved.