Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: a calorimetric study

Abstract

High-sensitivity isothermal titration calorimetry has been used to investigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 280.5-298 K. The present study unequivocally establishes the carbohydrate-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As-sB)2-type tetramer of the agglutinin has two equivalent sites that are non-interacting and independent. The site binding constants range from 2.2x10(3) M-1 at 282 K for galactose to 4.84x10(4) M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpies range from -21.9 kJ. mol-1 at 293 K for 4-methylumbelliferyl-beta-galactoside to -50.2 kJ. mol-1 at 292.9 K for thiodigalactoside. The observation of limited entropy-enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically driven. These studies show that the stronger binding of N-acetyl-lactosamine than lactose is due to a favourable interaction between the acetamido group of the reducing-end N-acetylglucosamine of the former and the corresponding loci in the agglutinin molecule. Preferential binding of methyl-beta-galactoside over methyl-alpha-galactoside also indicates the apolar nature of the interaction with the methyl group of the former sugar.

Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities

“…Figure 3a shows that for all probes 1-3, the probe concentration higher than 1 lM was required to successfully crosslink RCA, PNA, or ECA, which has the K d value of 37 lM, 323 lM, 400 lM, respectively. [12][13][14] The crosslinking yields did not reach saturation for all probes 1-3 for the concentration range studied, which can be explained by the fact that lectins were not fully bound by the probes below their corresponding K d values.…”

“…The binding affinity of each lectin toward D-lactose are in the order of RCA (K d = 37 lM) > ECA (K d = 323 lM) > PNA (K d = 400 lM). [12][13][14] In a typical PAL experiment, a photoaffinity probe and a lectin were incubated to form a binding complex at 0°C for 1 h then was irradiated at 365 nm at 0°C for an indicated duration of time. The reaction mixture was resolved by SDS-PAGE.…”

SPR Studies of Carbohydrate–Protein Interactions: Signal Enhancement of Low‐Molecular‐Mass Analytes by Organoplatinum(II)‐Labeling

“…Exploratory kinetic binding studies indicate that, as a consequence of the signal enhancement, values calculated for the PtCl(NCNÀ R)-labeled lactose/RCA 120 interaction (K D = 2 mm) are between 10 and 30 times higher than values previously determined for the lactose/RCA 120 interaction by isothermal titration calorimetry [37] or equilibrium dialysis calculation. [40,41] Discrepancies in K D values can also be observed between the PtCl(NCNÀR)-labeled mannose/Con A affinity measured by SPR (K D = 0.6 mm) and values obtained from titration microcalorimetry, [42] fluorescence anisotropy, [42] and SPR [19,43] for the methyl a-d-mannopyranoside/Con A interaction.…”

“…This observation is further supported by comparison of the SPR responses (at 1 mm concentration) of PtCl(NCNÀR)-labeled galactose 3 and Br(NCNÀR)-labeled lactose 9, possessing close molecular masses (M W = 745 vs. 755/757), but differing in the presence or absence of the platinum atom. Even though lactose has a higher affinity for RCA 120 than galactose, [37] the lack of the platinum atom in 9 causes a significant drop in RU relative to 3 (Figure 4). A curve relating the RU values at 26 mm concentration for lactose, 2, 9, 11, and 12 with their respective molecular masses ( Figure 5) reveals that the PtCl(NCNÀR)-labeled lactose 2 and the PtCl(NCNÀR)-labeled galactose 3 not only deviate from the linear trend presented by 9, 11, and 12, but also give responses corresponding to higher-molecular-mass compounds.…”

Thermodynamic investigations of ligand–protein interactions: Binding of the phenazinium dyes phenosafranin and safranin O with human serum albumin

“…This occurs generally with DC p o not equal to zero and DC p o ) DS o . This enthalpy-entropy compensation is generally associated with solvent reorganization accompanying protein-ligand interactions [40][41][42].…”