Fluorescence quenching and time-resolved fluorescence studies on Momordica charantia (bitter gourd) seed lectin.
Chemical modification studies implicated tryptophan (Trp) residues in the sugar binding activity of Momordica charantia lectin (MCL) [Mazumdar, T., Gaur, N. & Surolia, A. (1981) Eur. J. Biochem. 113, 463-470]. In the present study, the accessibility and environment of Trp residues in MCL were investigated by intrinsic fluorescence quenching and time-resolved fluorescence. The emission lamda max of native MCL in the absence as well as in the presence of 0.1 M lactose was around 335 nm, which shifted to 365 nm in the presence of 8 M urea, suggesting that the Trp residues which are predominantly buried in the hydrophobic core of the native lectin get exposed to the aqueous environment upon denaturation. At a quencher concentration of 0.5 M, the extent of quenching observed for the native MCL with acrylamide, I- and Cs+ was 46%, 17% and 12%, respectively. In the presence of 0.1 M lactose this quenching was smaller, suggesting that the sugar ligand provides a partial protection to the Trp residues. In time-resolved fluorescence measurements, the decay curves could be fitted well to a biexponential function with the estimated life times 0.92 ns and 4.64 ns for the native protein and 1.15 ns and 5.1 ns in the presence of 0.1 M lactose. All these results are consistent with the involvement of Trp residues in the sugar-binding activity of MCL.
Padma
P
P
School of Chemistry, University of Hyderabad, India.
Komath
S S
SS
Swamy
M J
MJ
D020106:Acrylamide; D002586:Cesium; D005453:Fluorescence; D007454:Iodides; D007785:Lactose; D037102:Lectins / Q000737:chemistry*; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D054790:Ribosome Inactivating Proteins, Type 2; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
D007454:Iodides; D037102:Lectins; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D002586:Cesium; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D009699:N-Glycosyl Hydrolases; D007785:Lactose