Measurement of the binding parameters of annexin derivative–erythrocyte membrane interactions

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca2+-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca2+-dependent binding that became partially Ca2+ independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein–membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein–Ca2+–membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV–Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2–2.5 mM Ca2+. We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.