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

Tzu Chen Yen, Shiaw Pyng Wey, Chang Hui Liao, Chi Hsiao Yeh, Duan Wen Shen, Samuel Achilefu, Tze Chein Wun

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)70-79
Number of pages10
JournalAnalytical Biochemistry
Volume406
Issue number1
DOIs
StatePublished - Nov 2010

Keywords

  • Annexin V
  • Erythrocyte
  • Fluorescein-5-isothiocyanate
  • Phosphatidylserine

Fingerprint

Dive into the research topics of 'Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions'. Together they form a unique fingerprint.

Cite this