Biomedical subjects

Ewa Pol

Publications and source records attributed to Ewa Pol.

4 recordsLinked to original sources

PubMed2006

Kinetic mechanism of deoxyadenosine kinase from Mycoplasma determined by surface plasmon resonance technology.

Surface plasmon resonance (SPR) detection technology was employed to investigate the kinetic mechanism of deoxyadenosine kinase from Mycoplasma mycoides ssp. mycoides SC. In our experimental approach, the enzyme was attached to the sensor surface, the reactants were injected in the mobile phase, and the product-enzyme complex formation was measured using the fact that the rate of product formation exceeds that of its dissociation. The pre-steady-state analysis of deoxyguanosine phosphorylation showed the presence of a burst phase, which is consistent with product dissociation being a rate-limiting step. High activity of the immobilized enzyme was demonstrated by analyzing the reaction mixture eluted from the chip and by determining the Michaelis-Menten constants for several phosphate acceptors (e.g., deoxyadenosine) and phosphate donors (e.g., ATP) using SPR detection. These values were in good agreement with those reported previously [Wang, L. et al. (2001) Mol. Microbiol. 42, 1065-1073]. The bisubstrate initial rate pattern obtained was characteristic of a sequential kinetic mechanism. Because in the method applied here it is the mass change on the surface that is monitored, a new mathematical approach to interpreting product inhibition experiments was proposed. According to that approach, product inhibition studies, supported by product binding experiments, indicated that the reaction mechanism was of Bi Bi sequential ordered type, involving the formation of a ternary complex, in which ATP and deoxyadenosine bound sequentially, followed by a transfer of the phosphate group, and an ordered release of products with ADP dissociating before dAMP.

Adenosine Diphosphate↗

PubMed2005

Analyzing a kinetic titration series using affinity biosensors.

The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call "kinetic titration." This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.

Antibodies, Monoclonal↗

PubMed2003

Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases.

The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.

Animals↗

PubMedDate not supplied

Biosensor-based characterization of serum antibodies during development of an anti-IgE immunotherapeutic against allergy and asthma.

Antibody responses, induced in Cynomolgus monkey by recombinant IgE-derived immunotherapeutic protein against atopic allergies and asthma, were characterized using label-free, real-time protein interaction analysis. The effects of two different immunotherapeutic proteins were compared. Active concentrations of specific anti-IgE antibodies formed were determined in sera sampled at multiple time points, using conditions of total mass transport limitation that were proved to exist on the sensor surface. These concentrations varied from about 0.4 to 35 microg/ml among the monkeys and throughout the immunization period. Based on these concentrations, the rate and affinity constants for the binding of antibody populations to the antigen could be determined. The apparent equilibrium dissociation constant decreased during the immunization period, for all the monkeys, by a factor between 6 and 50, ending at values from approximately 2 x 10(-9) to approximately 2 x 10(-11) M among the animals. This affinity maturation was attributable to the changes in both rate constants, although the magnitude of the contribution of each constant depended partly on specimen, but primarily on the immunotherapeutic used. The immunotherapeutic proteins examined showed excellent immunogenic properties, providing the basis for a new and effective treatment for allergy and asthma.

Animals↗