Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin.
Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.
Kenoth
Roopa
R
School of Chemistry, University of Hyderabad, India.
Swamy
Musti J
MJ
D005453:Fluorescence; D037102:Lectins / Q000737:chemistry*; D012639:Seeds / Q000737:chemistry*; D029778:Trichosanthes / Q000196:embryology*
D037102:Lectins