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).