Quantitative determination of dextran sulfate and pentosan polysulfate and their binding with protamine using...

We report for the first time a chronopotentiometric measurement of polyanions based on localized ion depletion at the sample/membrane interface at a characteristic transition time τ, using polymer membrane polyanion-selective electrodes. Chronopotentiometric transduction of polyions based on the measurement of transition time has analytically more attractive applications compared to the controlled-current reversible pulsed chronopotentiometric transduction based on electromotive force (emf) measurement. This is because traditional polyion-selective electrodes based on emf measurement intrinsically give nonlinear (sigmoidal) calibration curves. While these can be used for indirect determination of polyions via polyanion-polycation titrations, they are not convenient for direct quantitation. However, under chronopotentiometric measurement based on the measurement of transition time, the square root of the transition time τ is linearly related to the concentration of the polyion according to the Sand equation and can be used for a direct calibration-free rapid determination. In this work, we have measured the concentrations of dextran sulfate (DS) and pentosan polysulfate (PPS) using polyanion selective electrodes under chronopotentiometric method where the transition time was measured and controlled-current pulsed chronopotentiometric transductions, where the phase boundary potential (emf) was measured. In addition, the protamine-DS and the protamine-PPS binding ratios have been determined using both transductions. The protamine-PPS binding ratio was determined to be 1.51:1 by the titration method and 1.54:1 by chronopotentiometry. The protamine-DS binding ratio was determined to be 1.37:1 by the titration method and 1.41:1 by chronopotentiometry, showing excellent agreement between the two methods. These simple measurement methods of binding ratios between polysaccharides and polypeptides may become important tools for screening safer and more reliable antidotes for the newer and safer anticoagulants such as Low Molecular Weight Heparins(LMWHs) and also to determine the dosages of antidotes needed to neutralize the anticoagulant activity.