Imaging and Quantification of Secreted Peroxynitrite at the Cell Surface by a Streptavidin-Biotin-Controlled Binding Probe

Abstract

The ability to detect and image secreted peroxynitrite (ONOO^- ) along the extracellular surface of a single cell is biologically significant, as ONOO^- generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO^- , precise determination of the ONOO^- level at the cell surface remains a challenging task. In this paper, the use of a membrane-anchored streptavidin-biotin-controlled binding probe (CBP), ONOO-CBP, to determine quantitatively the ONOO^- level at the cell surface and to investigate the effect of different stimulants on the production of ONOO^- along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO^- secretion, achieving more than a 25-fold increase in ONOO^- relative to untreated cells. After 1 h of phorbol-12-myristate-13-acetate (PMA) stimulation, the amount of ONOO^- secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3-morpholinosydnonimine hydrochloride (SIN-1), which was estimated to release about 20 μm of ONOO^- into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods.

Keywords: biotin; cell surface; chemical probes; fluorescence; peroxynitrite.