Determination of low-molecular-weight heparins and their binding to protamine and a protamine analog using polyion-sensitive membrane electrodes

A polycation-sensitive membrane electrode based on the ion-exchanger dinony lnaphthalene sulfonate has previously been developed and used as an endpoin t detector for the determination of unfractionated heparin in whole blood s amples via simple potentiometric titration with protamine, Herein, we repor t the application of the same methodology for the quantitation of a commerc ial low-molecular-weight heparin (LMWH.) preparation (Fragmin) in whole blo od samples at concentrations up to 2 U/ml, Further, an analogous polyanion (heparin)-sensitive electrode is used to estimate the binding constants bet ween protamine and various LMWH preparations. The equilibrium constants (K- eq) and the number of binding sites per mole of heparin (n) are determined by recasting the data in the form of a Scatchard plot. Results show that th e average molecular weight and molecular weight distribution of the LMWH pr eparation are important parameters affecting their binding with protamine, Comparable binding constants are obtained for the same LMWH preparations ti trated with a synthetic protamine analog, [+18RGD] [acetyl-EA(R(2)A(2)R(2)A )(4)R(2)GRGDSPANH(2)]. (C) 1999 Academic Press.