Thermodynamic analysis of saccharide binding to snake gourd (Trichosanthes anguina) seed lectin

Abstract

The interaction of different saccharides with the snake gourd (Trichosanthes anguina) seed lectin (SGSL) was investigated by fluorescence spectroscopy. Binding of 4-methylumbelliferyl-b-d-galactopyranoside (MeUmbb Gal) to SGSL resulted in a significant increase in the fluorescence emission intensity of the sugar at 376 nm, and this change was used to estimate the association constants for the binding interaction. Interestingly, the increase in emission intensity changed with a change in temperature, increasing from 19.2% at 20 8C to 80.2% at 40 8C. At 20 8C the association constant, K a , for the MeUmbbGal±SGSL interaction was found by fluorescence titration to be 5.8 Â 10 4 m 21 . From the temperature dependence of the association constants, the changes in enthalpy (DH) and entropy (DS) associated with binding of MeUmbbGal to SGSL were estimated to be 280.85 kJ´mol 21 and 2184.0 J´mol 21´K21 , respectively. Binding of unlabeled sugars was investigated by monitoring the decrease in fluorescence intensity when they were added to a mixture of SGSL and MeUmbbGal. The K a values for different sugars were determined at several temperatures, and DH and DS were determined from the van't Hoff plots. Enthalpy±entropy compensation was noticed in all cases. The results indicate that saccharide binding to SGSL is enthalpy-driven and the negative contribution from entropy is, in general, quite high.Keywords: agglutinin; enthalpy±entropy compensation; fluorescence spectroscopy; 4-methylumbelliferyl-b-dgalactopyranoside; thermodynamic analysis.The extraordinary diversity in their recognition of carbohydrate structures and their ubiquitous distribution in the plant and animal kingdoms make lectins a very interesting and unusual group of proteins [1]. To date, a large number have been isolated from different plant sources, and in terms of sheer numbers they greatly outweigh lectins from other sources such as viruses, bacteria, fungi or animals [2]. Plant lectins are used extensively in purification and structural characterization of glycoconjugates, investigation of cell-surface architecture, blood typing, and fractionation of cells [3,4].As a variety of biological recognition processes are mediated via lectins or lectin-like molecules and their corresponding receptors, the study of lectin±sugar interactions is of utmost importance in the characterization of any new lectin [1]. Such information is essential for assessing the affinity of a protein for a given ligand. A knowledge of the differences in binding affinity of a lectin for different sugars helps to define the architecture of the combining site of the lectin and identify the functional groups of the sugar that make positive contributions to the interaction (for examples, see [5,6]). Further, thermodynamic studies can reveal the effect of increasing the carbohydrate chain length/branching and the effect of substituents on lectin recognition [7,8]. Given the strength and specificity of the interaction, lectin±carbohydrate binding studies also provide ideal models for stu...

Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin

“…These values correspond to approx. 30.7% β-sheet, 17.4% β-turn and 39% random coil. Prediction by DPM gave 14, 37, 15 and 34 percentages for α-helix, β-sheet, β-turn and random coil.…”

“…These include MCL (Momordica charantia lectin) and Trichosanthes kirilowii lectin from the seeds and LAA (Luffa acutangula agglutinin) and CIA (Coccinia indica agglutinin) from the phloem exudate of cucurbits [7][8][9][10][11]. In order to characterize the cucurbit seed lectins in greater detail, we initiated a long-term programme in the mid-1990s and reported on the purification, physicochemical characterization and carbohydrate-binding specificity of three new lectins from the seeds of Cucurbitaceae species, namely SGSL [Trichosanthes anguina (snake gourd) seed lectin], TCSL (Trichosanthes cucumerina seed lectin) and TDSL (Trichosanthes dioica seed lectin), and characterized the carbohydrate-binding properties of MCL in greater detail [12][13][14][15][16][17][18][19][20][21][22][23][24]. Additionally, the interactions of several water-soluble porphyrins with SGSL, TCSL and MCL were also investigated [25][26][27].…”

Spectroscopic and differential scanning calorimetric studies on the unfolding of Trichosanthes dioica seed lectin. Similar modes of thermal and chemical denaturation

“…This is somewhat similar to the activity of other Cucurbitaceae seed lectins. MCL and TCSL exhibits maximum activity in the pH range from 8.0-11.0 [14,15], whereas another cucurbit seed lectin, SGSL exhibits maximum activity in the pH range of 6.0-10.0 [17]. The data presented in Fig.…”

“…In comparison, the activity of TCSL was unaffected up to about 60°C, and decreased to 50% at 70°C, indicating that it is thermally more stable than TDSL. However, TDSL appears to be more stable than MCL and SGSL, which were inactivated by incubation above 50°C and 40°C, respectively [14,15,17]. It is interesting to note that while the DSC and CD studies indicate that TDSL undergoes thermal unfolding in the temperature range of 65-70°C, the activity studies on samples incubated at different temperatures show that the lectin activity starts to decrease from 60°C onwards.…”

“…Studies on lectins from other families are fewer, and there is a need to characterize them in detail to understand their properties in detail. Our laboratory has been working on the purification and characterization of lectins from the Cucurbitaceae with respect to physicochemical properties, carbohydrate binding and hydrophobic ligand binding [13][14][15][16][17][18].…”