Atomic Force Microscopy and Quartz Crystal Microbalance Study of the Lectin-Carbohydrate Interaction Kinetics

Abstract

Two analytical methods, atomic force microscopy and quartz crystal microbalance, were applied to the study of the reaction kinetics occurring between concanavalin A and carboxypeptidase Y, presenting the specific lectin-carbohydrate recognition. The dissociation rate constants for concanavalin A-carboxypeptidase Y complex obtained using both atomic force microscopy and quartz crystal microbalance were of the same order of magnitude: k diss = 0.170 ± 0.060 s −1 and k diss = 0.095 ± 0.002 s −1 , respectively. In addition, each method alone aided in determining other parameters characterizing the studied interaction. Quartz crystal microbalance permitted us to estimate the association rate (k ass = (5.6 ± 0.1) × 10 4 M −1 s −1 ) and the equilibrium (Ka = (0.59 ± 0.01) × 10 6 M −1 ) constants for the binding process occurring between concanavalin A and mannose residues of carboxypeptidase Y under given experimental conditions. Atomic force microscopy in force spectroscopy mode enabled the determination of the energy barrier position of r = 2.29 ± 0.04Å characterizing the dissociation of concanavalin Acarboxypeptidase Y molecular complex. The presented results show that both atomic force microscopy and quartz crystal microbalance can be used to determine quantitative parameters characterizing the specific molecular interaction. Both methods can be easily combined for complementary and/or alternative studies of a chosen molecular interaction. By preparing the samples in the same manner the direct comparison between the data obtained via atomic force microscopy and quartz crystal microbalance can be made.

Investigating the Effect of Tyrosine Kinase Inhibitors on the Interaction between Human Serum Albumin by Atomic Force Microscopy

“…The gathered k off values for HSA complexes ranged from 0.106 s −1 (control conditions) to 0.256 s −1 (in the presence of imatinib inhibitor). These data are in agreement with previous reported biological systems such as ferredoxin NADP + reductase: NADP + ( k off = 0.105 s −1 ) [ 50 ]; aggrecan: aggrecan ( k off = 0.127 s −1 ) [ 51 ]; concavalin A: carboxipeptidase A ( k off = 0.170 s −1 ) [ 52 ]; or lectin: α-GalNAc ( k off = 0.680 s −1 ) [ 53 ]. This finding evidences the transient nature of the bonds involved in HSA complex under the five studied conditions.…”

Influence of Temperature on the Nanoadhesion of a Methyl-Terminated Thiol Monolayer: A New Insight with High-Rate Dynamic Force Spectroscopy

“…However, none of these modelsincluding the DHS modelcan explain directly the 2-fold character (regimes I and II) of the increase. For specific bonds, the protein–ligand interaction, for instance, an interpretation has been developed that explains the two regimes as due to two barriers: one outer (higher) responsible for regime I and one inner responsible for regime II. , The latter is revealed at high loading rates because then the outer barrier is strongly lowered by external force at the moment of forced unbinding. However, this interpretation cannot be applied to the case of nonspecific, mainly van der Waals, interactions, which are characterized by a Lennard-Jones type of potential.…”

Concanavalin A–Polysaccharides binding affinity analysis using a quartz crystal microbalance

“…The binding of Con A to mannose and glucose containing oligosaccharides has been intensively studied and to date, several carbohydrate binding constants to Con A are known (Dam et al, 2000; Duverger et al, 2003; GLEW, 1973; K. Lebed, 2007).…”