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Binding of 4‐methylumbelliferyl β‐ d ‐galactopyranoside to Momordica charantia Lectin

1981; Wiley; Volume: 113; Issue: 3 Linguagem: Inglês

10.1111/j.1432-1033.1981.tb05087.x

1432-1033

M.I. Khan, Tripti MAZUMDER, Debkumar Pain, Namita Gaur, Avadhesha Surolia,

Protein Interaction Studies and Fluorescence Analysis

The binding of 4‐methylumbelliferyl β‐ d ‐galactopyranoside (MeUmb‐Gal p ), to Mormordica charantia lectin was studied by equilibrium dialysis and quenching of ligand fluorescence. The fluorescence of MeUmb‐Gal p decreases as a function of solvent polarity. On binding to M. charantia lectin, its fluorescence was nearly 100% quenched, showing that the binding of the glycoside takes place in a hydrophobic environment. The binding of the fluorescent sugar was saccharide‐specific as evidenced by reversal of MeUmb‐Gal p fluorescence quenching by lactose. The association constant is independent of the experimental method used and at 25° C the value is (1.96 ± 0.05) × 10 4 M −1 . The number of binding sites as determined by equilibrium dialysis and fluorescence quenching agree very well with each other; n being equal to 1.98 ± 0.02. The k a value for the glycoside was also determined by competition studies employing reversal of fluorescence quenching of MeUmb‐Gal p by lactose. The value of K a obtained for lactose is 1.21 × 10 4 M −1 at 30° C. The internal consistency of the association constant and number of binding site values at low and high saturation indicates the absence of additional subsite on M. charantia lectin. The thermodynamic parameters do not differ greatly with change in temperature: the values of −Δ H ° and −Δ S ° are equal to 30 ± 0.63 kJ mol −1 and 21 ± 0.3 J mol −1 K −1 respectively in the range of 15–35° C indicating that the binding of M. charantia lectin to saccharide is exothermic in nature.