Reaction pathway and free energy profile determined for specific recognition of oligosaccharide moiety of carboxypeptidase Y.

The interaction of mannose specific lectin (from Lens culinaris, LcL) with the carbohydrate moiety of carboxypeptidase Y (CaY) was studied using both atomic force microscope (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D). The AFM enables to determine the positions of energy barriers present in the energy landscape of the single complex undergoing dissociation. The QCM-D measurements allow the estimation of the quantitative parameters characterizing the kinetics of the studied molecular interaction (namely the association and dissociation rate constants and the association constant). The use of both methods not only delivers the complementary characterization of kinetic and thermodynamic parameters but also permits to investigate the mechanism of the binding and unbinding of the molecules. The results for LcL were compared with those obtained for concanavalin A i.e. lectin, which interacts with the carbohydrate moiety on a similar way.

Erythrocyte stiffness in diabetes mellitus studied with atomic force microscope.

The mechanical properties of erythrocyte membrane have been studied using an atomic force microscope. Measurements were carried out on blood samples taken from 7 diabetes mellitus patients and 8 healthy individuals. For each blood sample a distribution of a Young's modulus was constructed. It has been found that both the mean value and the width of the distribution in diabetic patients exceed the corresponding results for healthy persons by a factor greater than 3. The high sensitivity of the atomic force microscopy and the ability to measure the full distribution of the erythrocyte membrane Young's modulus makes it a unique, powerful and promising tool in studies of the membrane stiffness of red blood cells.

Lectin-carbohydrate affinity measured using a quartz crystal microbalance.

The association of two molecules is described by two parameters, association equilibrium and association rate constants, which are characteristic for a given type of interaction. Usually, they are determined for interacting molecules dissolved in solution. However, for many applications one type of molecules is immobilized on a substrate, which may influence the binding kinetics. The studied complex of concanavalin A and carboxypeptidase Y belongs to the lectin-carbohydrate type of interaction involving the recognition of oligosaccharide moieties. The concanavalin A was immobilized on a gold electrode of quartz crystal, while carboxypeptidase Y was added to a buffer (Tris-buffered saline). The constants describing the association of the investigated molecules were determined on the basis of measurements performed using a quartz crystal microbalance in liquid. The obtained values were (0.59+/-0.01)x10(6) M(-1) for the association equilibrium constant and (5.6+/-0.1)x10(4) M(-1)s(-1) for the association rate constant. The saturation binding experiment gave another value of the association constant, (2.7+/-0.02)x10(6) M(-1). The comparison of obtained values with previously published ones verifies that the molecule orientation and binding site accessibility for specific ligands could influence the association equilibrium constant value. The presented measurements demonstrate the ability of a quartz crystal microbalance to detect and to evaluate the association process occurring between molecules.

Erythrocyte stiffness probed using atomic force microscope.

The stiffness of erythrocytes in patients (N=45) suffering from certain disorders, such as coronary disease, hypertension, and diabetes mellitus has been assessed using the Atomic Force Microscopy (AFM) and compared with that in a group of healthy individuals (N=13). For each blood sample, around 20 erythrocytes were selected at random and the stiffness of each one was probed in 20-30 arbitrarily chosen points. From these results, distributions of the cell Young's modulus (YM) were determined. Average values and widths of YM distributions significantly increased in samples taken from diabetes mellitus patients and cigarette smokers, as compared to those taken from healthy donors. At the same time, the average values of YM were found to increase as a function of the patient's age. We demonstrated that the atomic force microscope is a very sensitive tool for determination of cell stiffness with every prospect of a routine application as a diagnostic tool in quantitative analysis of the physiological and pathological states of red blood cells.