Jean-Maxime Nigretto

Contributions of negatively charged chemical groups to the surface-dependent activation of human plasma by soluble dextran derivatives

Negatively charged surfaces are known to promote contact activation. The mechanism responsible for increasing affinity for surfaces is not yet quite understood, although the presence of negative charge densities is thought to be a prerequisite. With the availability of soluble dextran derivatives, varyingly substituted with charged methylcarboxylate, methylbenzylamide sulphonate and uncharged methylbenzylamide residues, we were able to discriminate between the contributions of these chemical moieties to contact activation, thus suggesting that the stimulating properties of synthetic negatively charged surfaces should also be described in terms of specific interactions instead of global negative charge density. This could be effected by quantifying the activating capacities as a function of the chemical group composition. A direct correlation linking activating capacities to anticoagulant properties has been observed.

Potential- and surface-dependent behaviour of thrombin adsorbed on carbon electrodes

A procedure is described for the direct immobilization of thrombin on carbon paste and solid carbon electrodes with good reproducibility and improved stability for the enzyme. Typically, the radiolabelling of thrombin with iodine-125 indicated that enzyme densities of about 2 × 10−13 mol cm−2 could be achieved at saturation. This gave a rate of coverage of about 20% of the electrochemical area. The catalytic activities and the kinetic parameters were estimated under forced convection conditions with a specific substrate (S-2238) provided with chromogenic properties and dissolved in the solution. No major changes in Km or Vm were found to be adsorption. However, increased activities were detected when adsorption was conducted at positive prepolarization of the electrode or after potential cycling in the presence of p-nitroaniline. A pattern of interaction with the surface sites of adsorption is proposed.

Effect of surface activation on charge and mass transfer rates of the hexacyanoferrate(III)/(II) redox probe at fibrinogen-modified carbon paste electrodes

Abstract The electron transfer and mass transport properties of fibrinogen-modified carbon paste surfaces, following preliminary activation for various duration, has been studied using cyclic voltammetry (CV) and chronoamperomety. Activation was carried out by subjecting the surface, exposed to a solution-containing oxidant species, to positive polarization of increasing duration. Modification was subsequently achieved following spontaneous adsorption of fibrinogen on exposure to a solution containing the protein. To investigate the electrochemical properties of this surface, the variations in the charge-transfer and mass-transport kinetics of hexacyanoferrate(III)/(II) ions, used as a redox probe, were analyzed separately. The results showed the favorable effect of activation on electrocatalysis and charge-transport properties through the coating. Relative to pretreated bare surfaces, however, the presence of fibrinogen induced opposed variations, thus explaining unchanged CV peak currents. These features were essentially attributed to morphological changes promoted by activation in the protein layer.

Electrochemical assay and properties of plasmin adsorbed onto a carbon paste electrode

Abstract In this report, we describe the catalytic properties of plasmin (Pln) adsorbed irreversibly onto a carbon paste electrode via its surface reaction with an electrogenic substrate (S-2644) synthesized specially for this purpose. Results show that the surface properties of Pln were retained in the potential range required for the electrochemical assay and under optimal conditions (pH 8.5 and 5 °C). Experimental data were linearized by using Lineweaver-Burk plots. The feasibility and the reliability of the assay was demonstrated from the linear relationships obtained with two different Pln-modified surfaces which led us to deduce the apparent Michaelis constant related to the catalysis. Homogeneous and heterogeneous values for Km obtained using parallel photometrical and electrochemical assays under various conditions are given.

Electrochemical activation of human factor XII (Hageman factor) immobilized on carbon electrodes

Abstract Direct potential-mediated activation of purified human factor XII (FXII) immobilized on carbon electrodes has been evidenced. This required first to find a procedure for the immobilization of FXII on the electrode with good reproducibility. This adsorption isotherms of this zymogen and those of the enzymatic form, FXIIa, were characterized and dicussed. The catalytic activities were estimated under stirring with specific substrate (S-2302) provided with chromogenic properties and dissolved in the solution. Pretreatment of the electrode with negative square-wave potential pulses resulted in changes in the adsorption capacity of the carbon surface towards FXIIa. When the untreated electrode was covered with FXII at saturation, application of these pulses generated catalytic activities according to first-order kinetics.