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, 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.
Yen
Tzu-Chen
TC
Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Chang Gung University School of Medicine, Taoyuan, Taiwan.
Wey
Shiaw-Pyng
SP
Liao
Chang-Hui
CH
Yeh
Chi-Hsiao
CH
Shen
Duan-Wen
DW
Achilefu
Samuel
S
Wun
Tze-Chein
TC
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D011310:Preservatives, Pharmaceutical / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
C587037:ANV-6L15 protein; D017304:Annexin A5; D011310:Preservatives, Pharmaceutical; D011993:Recombinant Fusion Proteins; D004492:Edetic Acid; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium