Fluorescence and circular dichroism studies on the accessibility of tryptophan residues and unfolding of a jacalin-related α-D-galactose-specific lectin from mulberry (Morus indica)

MLGL (Mulberry Latex Galactose-specific Lectin) is an alpha-D-galactose binding lectin isolated from the latex of mulberry (Mores indica) tree and contains two tryptophan residues in each of its subunits. The fluorescence emission maximum of native MLGL seen at 326 nm shifts to 350 nm upon incubation with 6 M guanidinium thiocyanate (Gdn.SCN), suggesting that the tryptophans are located inside the hydrophobic core of the protein and become fully exposed upon denaturation. Fluorescence quenching studies revealed that the neutral acrylamide exhibits the highest quenching, with similar to 33% of total fluorescence in the native protein being quenched at a quencher concentration of 0.5 M, whereas iodide (similar to 24%) and cesium (similar to 4%) ions showed significantly lower quenching. With the denatured protein, acrylamide quenching involves both dynamic and static processes as evident from an upward curving Stern-Volmer plot. Time-resolved fluorescence studies showed two lifetime components of 3.7 ns and 1.3 ns for the native protein, while three lifetime components were observed for the denatured protein. MLGL showed high resistance to urea (up to 8 M) and guanidine hydrochloride (up to 6 M), whereas treatment with 6 M Gdn.SCN completely denatured the protein, via a broad sigmoidal transition with a transition midpoint at similar to 3.75 M. Circular dichroism studies and hemagglutination assays showed that the secondary and tertiary structures as well as lectin activity of MLGL were unaffected up to 70 degrees C. Additionally, pH dependent studies showed that the secondary structure of MLGL is unaltered in the pH range 6.2 to 8.5, but a decrease in lectin activity is observed (similar to 50%) at pH 6.2.