Impacts of Papain and Neuraminidase Enzyme Treatment on Electrohydrodynamics and IgG-Mediated Agglutination of Type A Red Blood Cells

Abstract

The stability of native and enzyme-treated human red blood cells of type A (Rh D positive) against agglutination is investigated under conditions where it is mediated by immunoglobuline G (IgG) anti-D antibody binding. The propensity of cells to agglutinate is related to their interphasic (electrokinetic) properties. These properties significantly depend on the concentration of proteolytic papain enzyme and protease-free neuraminidase enzyme that the cells are exposed to. The analysis is based on the interpretation of electrophoretic data of cells by means of the numerical theory for the electrokinetics of soft (bio)particles. A significant reduction of the hydrodynamic permeability of the external soft glycoprotein layer of the cells is reported under the action of papain. This reflects a significant decrease in soft surface layer thickness and a loss in cell surface integrity/rigidity, as confirmed by nanomechanical AFM analysis. Neuraminidase action leads to an important decrease in the interphase charge density by removing sialic acids from the cell soft surface layer. This is accompanied by hydrodynamic softness modulations less significant than those observed for papain-treated cells. On the basis of these electrohydrodynamic characteristics, the overall interaction potential profiles between two native cells and two enzyme-treated cells are derived as a function of the soft surface layer thickness in the Debye-Hückel limit that is valid for cell suspensions under physiological conditions (approximately 0.16 M). The thermodynamic computation of cell suspension stability against IgG-mediated agglutination then reveals that a decrease in the cell surface layer thickness is more favorable than a decrease in interphase charge density for inducing agglutination. This is experimentally confirmed by agglutination data collected for papain- and neuraminidase-treated cells.

Impact of the virulence-associated MAb3/1 epitope on the physicochemical surface properties of Legionella pneumophila sg1: An issue to explain infection potential?

“…(1)-(7) disregard (i) polarization of electric double layer by the applied field and (ii) the possible heterogeneity in soft material density distribution within the permeable part of the bacteria, heterogeneity occasioned by possible swelling of the soft bacterial component under low ionic-strength conditions. As extensively demonstrated in previous work [33,36,37], phenomena (i) and (ii) effectively lead to reduced mobility (in magnitude) as compared to those obtained from Eqs. (1)- (7).…”

“…For the sake of illustration, recent analysis demonstrated that electrokinetics is a fast and valuable tool for detecting surface structure modifications/alterations undergone by soft bioparticles like blood cells or yeasts as a result of the action of biospecific molecules like e.g. enzymes [37,38]. In the current study, electrophoretic mobilities measured as a function of KNO 3 solution ionic strength were quantitatively interpreted on the basis of the analytical theory developed by Ohshima which accounts for the electrokinetic response of soft cylindrical particles [39].…”

Theory of electrostatics and electrokinetics of soft particles

“…The diffuse character of the polyelectrolyte layer is defined by a gradual distribution of the density of polymer segments in the interspatial region separating the core from the bulk electrolyte solution. Successful application of that model has been reported for humic acids [46], bacteria [47] and red blood cells [48]. For numerical calculations of the electrophoretic mobility based on more rigorous theories, the readers are referred to the studies of Hill et al [49][50][51][52] and Lopez-Garcia et al [53][54][55] as well as that of Duval and Ohshima [45].…”

On the applicability of the Brinkman equation in soft surface electrokinetics

“…This means that the mean field approximation fails on the periphery of diffuse polyelectrolyte layers where the density of charged segments is lower than a given critical value. Despite this difficulty and the underlying limitations of the model, failure of the mean-field approach for electrostatics in the very diffuse portion of the gel layer is not fatal because previous experimental and theoretical publications on numerous (bio)colloidal systems [58][59][60] demonstrated that the electrokinetics of soft diffuse interfaces is for a large part dominated by the hydrodynamics and not by the electrostatics of the tails of charged segments pointing toward the solution. Rigorously, this means, that a correction term should be added to the governing electrostatic equation and that the impact of this term on the overall electrokinetic response of the system should turn out to be small and thus could be possibly disregarded.…”