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

The interaction of different saccharides with the snake gourd (Trichosanthes anguina) seed lectin (SGSL) was investigated by fluorescence spectroscopy. Binding of 4-methylumbelliferyl-β-d-galactopyranoside (MeUmbβGal) 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 °C to 80.2% at 40 °C. At 20 °C the association constant, Ka, for the MeUmbβGal–SGSL interaction was found by fluorescence titration to be 5.8 × 10⁴ m⁻¹. From the temperature dependence of the association constants, the changes in enthalpy (ΔH) and entropy (ΔS) associated with binding of MeUmbβGal to SGSL were estimated to be −80.85 kJ·mol⁻¹ and −184.0 J·mol⁻¹·K⁻¹, respectively. Binding of unlabeled sugars was investigated by monitoring the decrease in fluorescence intensity when they were added to a mixture of SGSL and MeUmbβGal. The K_a values for different sugars were determined at several temperatures, and ΔH and ΔS 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

Physicochemical and saccharide-binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina

Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0–11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 °C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% β-sheet, 10.6% β-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the α-helix content is negligible. Binding of 4-methylumbelliferyl-β-d-galactopyranoside (MeUmbβGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 °C, the association constant, Ka, for the TCSL–MeUmbβGal interaction was determined as 6.9x104M-1. Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbβGal was titrated with TCSL

Role of histidine residues in the sugar-binding activity of Trichosanthes cucumerina seed lectin

Chemical modification studies with group specific reagents indicated that the imidazole side chains of histidine residues are involved in the carbohydrate-binding activity of Trichosanthes cucumerina seed lectin (TCSL). A total of 9.8 (±1.8) histidine residues could be modified by reaction with diethyl pyrocarbonate when the reaction was carried out for 2 hours, which resulted in a total loss in the carbohydrate binding and hemagglutinating activities of the lectin. Reversing the modification by treating the histidine-modified protein with hydroxylamine resulted in a complete recovery of the activity. Presence of the specific sugar (0.2 M galactose) afforded a partial protection in that only 4.6 His residues could be modified in the same period of time. In the presence of 6 M guanidinium hydrochloride, 15.8 (±1.5) His residues were modified. The histidine-modified lectin cross-reacted with rabbit anti-TCSL antiserum, indicating that the conformation of the modified lectin is unaltered and that the loss of activity is not a consequence of structural changes. Modification of the side chains of lysine, tyrosine and cysteine residues did not affect the haemagglutination activity of the lectin. The tryptophan residues of native TCSL could not be modified by N-bromosuccinimide. However, modification with this reagent in the presence of 8 M urea indicated that there are 4.6 (±0.4) Trp residues in the dimeric lectin

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7×104-6.2×105 M-1, with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins

N-Myristoylethanolamine-cholesterol (1:1) complex: first evidence from differential scanning calorimetry, fast-atom-bombardment mass spectrometry and computational modelling

The interaction of N-myristoylethanolamine (NMEA) with cholesterol is investigated by differential scanning calorimetry (DSC), fast-atom-bombardment mass spectrometry (FAB-MS) and computational modelling. Addition of cholesterol to NMEA leads to a new phase transition at 55°C besides the chain-melting transition of NMEA at 72.5°C. The enthalpy of the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, vanishing at 80 mol%. The enthalpy of the chain-melting transition of NMEA decreases with an increase in cholesterol; the transition disappears at 50 mol%. FAB-MS spectra of mixtures of NMEA and cholesterol provide clear signatures of the formation of {[NMEA+cholesterol]+} {[NMEA+cholesterol+Na]+}. These results are consistent with the formation of a 1:1 complex between NMEA and cholesterol. Molecular modelling studies support this experimental finding and provide a plausible structural model for the complex, which highlights multiple H-bond interactions between the hydroxy group of cholesterol and the hydroxy and carbonyl groups of NMEA besides appreciable dispersion interaction between the hydrocarbon domains of the two molecules