One-step isocratic high-performance liquid chromatographic purification of radioiodinated and radioiodinated-photoactivable derivatives of cholecystokinin.

N-Hydroxysuccinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate (the Bolton-Hunter reagent) was conjugated with (Thr 34, NLeu 37) cholecystokinin (CCK) 31-39 in anhydrous dimethylformamide-pyridine in 20% yield. The radiolabelled peptide was purified from the reaction mixture in one step, by isocratic elution from a C18 high-performance liquid chromatographic (HPLC) column with 35% aqueous acetonitrile-0.13% heptafluorobutyric acid as eluent. The concentration of the radiolabelled peptide was estimated by UV monitoring. The acylating photoactivable radioiodinated reagent N-hydroxysuccinimidyl-N-(4-azido-2-nitrophenyl)-3-[125I]iodotyrosi ne was synthesized, purified on a C18 HPLC column by isocratic elution with 65% acetonitrile-1 mM hydrochloric acid, then conjugated with (Thr 34, NLeu 37) CCK31-39. The resulting photoactivable radioiodinated CCK analogue was purified by isocratic elution on a C18 HPLC column with 39% aqueous acetonitrile-0.1 M triethylamine phosphate (pH 3.5). The binding ability of both tracers and their non-radioactive analogues to CCK receptors was tested on rat pancreatic plasma membranes. As compared to a KD of 4.5 nM for unmodified (Thr 34, NLeu 37) CCK31-39, the KD of the radioiodinated Bolton-Hunter derivative was 3 nM, and that of the photoactivable radioiodinated derivative was 19 nM.