Quantitative analysis of annexin V–membrane interaction by flow cytometry

• DOI: 10.1007/s00249-015-1026-9
• Publication Date: 2015-04-28T04:57:38Z
• Authors: Jie Wang; Liangqiang He; Dianhua Chen; Yazhou Pi; Wenping Zhou; Xingkui Xiong; Yongzhe Ren; Yueyang Lai; Zichun Hua
• Keywords: 0303 health sciences; 03 medical and health sciences; Cell Membrane; Humans; Calcium; Annexin A5; Models, Theoretical; Flow Cytometry; Protein Binding

We constructed a green fluorescent phosphatidylserine (PS)-binding probe, which was generated by fusing enhanced green fluorescent protein (EGFP) to the C terminus of human annexin V (anxV). With this probe, we investigated anxV-membrane interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM). A mathematical description of the binding characteristics is proposed and validated to quantify the relationship concerning the relative concentration of membrane-bound anxV (B), calcium concentration ([C]), and protein concentration ([P]). Further analyses reveal that [Formula: see text] is linear with [Formula: see text] or [Formula: see text] when [P] and [C] are fixed, respectively, which indicates that the anxV-membrane binding reaction may involve sequential multiple steps. Our study provides a reference for application of anxV in apoptosis detection. The mathematical expression facilitates exploration of the possible interactions between calcium, anxV, and membrane. The corresponding mathematical analysis strengthens the interpretation of the interaction data.