Oxidative iodination
The radioligands were created by oxidative iodination with the oxidizing agent ChloramineT. Here the iodine isotope [125I] becomes incorporated in the Tyr residue at position 10 of hGLP-2(1-33,M10Y) or hGLP-2(3-33,M10Y). 2nmol peptide was dissolved in 10µL iodination buffer (100 mM phosphate buffer) and 0.4 nCi Na125I was added. A stepwise, so-called stoichiometric oxidation reaction was performed by sequential addition of 6 aliquots of 5 µL ChloraminT (30 µg/mL) with 1 min intervals during constant stirring. Under these conditions [125I] is incorporated at the hydroxyl group in the ortho position of the Tyr residue. The reaction was terminated by the addition of 400 µL phase A (0,1% trifluoracetic acid (TFA)). The reaction was carried out in at pH 7.4 to avoid labeling of histidine residues at basic conditions (pH > 8.5). The product was fractionated on a high-performance liquid chromatography (HPLC) (Åkta, GE Healthcare, Boston US) with a C18 column for reverse-phase (RP) HPCL (RP-HPCL). The column was initially flushed with 80% phase A and 20% phase B (Acetonitrile + 0,1% TFA), and terminally by applying increasing concentrations of phase B. The pressure of the RP-HPCL was kept constant at 8 MPa with a flow of 1 mL/min. Before binding assays were performed the eluted fractions were tested in homologous competition binding (see next section for method).