Submitted on February 15, 2005
Revised on March 17, 2005
Accepted on March 24, 2005
Fluorescent leukotriene B4: potential applications
Alan Sabirsh, Anders Wetterholm, Jesper Bristulf, Hakon Leffler, Jesper Z. Haeggstrom, and Christer Owman
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177
Corresponding Author: Alan.Sabirsh{at}mbb.ki.se
Leukotriene B{super4} (LTB{super4}) is a potent lipid mediator of inflammation, which acts primarily via a seven-transmembrane spanning, G-protein coupled, receptor denoted BLT{sub1}. Here we describe the synthesis and characterization of fluorescent analogues of LTB{super4} which are easy to produce, inexpensive and without the disadvantages of a radioligand. Fluorescent LTB{super4} is useful for labelling LTB{super4} receptors for which no antibodies are available and, for performing one-step fluorescence polarisation assays conducive to high-throughput screening. We found that orange and green fluorescent LTB{super4} were full agonists that activated the LTB{super4} receptor BLT{sub1} with EC50s of 68 and 40 nM respectively (4.5 nM for unmodified LTB{super4}). Flow cytometric measurements and confocal imaging showed that fluorescent LTB{super4} co-localised with BLT{sub1}. Fluorescence polarisation measurements showed that orange fluorescent LTB{super4} bound to BLT{sub1} with a Kd of 66 nM, and that this binding could be displaced by unlabelled LTB{super4}, and other BLT{sub1} specific ligands. Fluorescent LTB{super4} analogs were also able to displace tritiated LTB{super4}. Orange fluorescent LTB{super4} binding to EGFP-tagged BLT{sub1} could be observed using fluorescence resonance energy transfer (FRET). In addition to being a useful alternative to radiolabelled LTB{super4}, the unique properties of fluorescently labelled LTB{super4} allow a variety of detection technologies to be employed.
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