REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF RADIOIODINATED SALMON CALCITONINS

Reversed-phase HPLC conditions for simultaneous separation of salmon c alcitonin, mono- and di-radioiodinated salmon calcitonins and their tr yptic digested fragments have been developed. Salmon calcitonin was ra dioiodinated with (NaI)-I-125 by the iodo-beads method. After solid-ph ase extraction from the reaction mixtures using C-18 Bond Elut cartrid ges, mono- and di-radioiodinated salmon calcitonins were separated fro m each other, as well as from unlabeled salmon calcitonin, on a Bondcl one 10 C-18 column (300 x 7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evalu ated on the basis of UV absorbance (215 and 280 nm), fluorescence (lam bda(ex) = 282 nm, lambda(em) = 310 nm) and measurement of specific rad ioactivity by means of a flow-through radio-isotope detector. HPLC sep aration of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C-18 300 Angstrom column (250 x 4.6 mm I.D.) and subseque nt amino acid analysis, led to the conclusion that radioiodination too k place at the Tyr residue and not at the His moiety.