Radiolabeled annexin V (ANV) has been widely used for imaging cell apoptosis. Recently, a novel ANV-Kunitz-type protease inhibitor fusion protein, ANV-6L15, was found to be a promising probe for improved apoptosis detection based on its higher affinity to phosphatidylserine (PS) compared to native ANV. The present paper investigates the feasibility of apoptosis detection using radioiodinated ANV-6L15. Native ANV and ANV-6L15 were labeled with iodine-123 and iodine-125 using Iodogen method. The binding between the radioiodinated proteins and erythrocyte ghosts or chemical-induced apoptotic cells was examined. ANV-6L15 can be radioiodinated with high yield (40%-60%) and excellent radiochemical purity (>95%). (123)I-ANV-6L15 exhibited a higher binding ratio to erythrocyte ghosts and apoptotic cells compared to (123)I-ANV. The biodistribution of (123)I-ANV-6L15 in mice was also characterized. (123)I-ANV-6L15 was rapidly cleared from the blood. High uptake in the liver and the kidneys may limit the evaluation of apoptosis in abdominal regions. Our data suggest that radiolabeled ANV-6L15 may be a better scintigraphic tracer than native ANV for apoptosis detection.
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, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) 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-Ca(2+)-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 Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.
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