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 1.2-2.5mM 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.
- Annexin V