A14-[125I]monoiodoinsulin purified by different high-performance liquid chromatographic procedures and by polyacrylamide gel electrophoresis: preparation, immunochemical properties and receptor binding affinity

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation of A14-[125I]monoiodoinsulin directly from the iodination mixtures. It remains to be clarified, however, whether the RP-HPLC chromatographic conditions affect the properties of the purified tracer. In this study we prepared A14-[125I]insulin purified by polyacrylamide gel electrophoresis (PAGE) and by three different RP-HPLC mobile phases containing, respectively, ammonium acetate, sodium perchlorate and trifluoroacetic acid. The binding characteristics of all these tracers were examined using an insulin antiserum and insulin cell receptors. The specific radioactivity corresponded to the theoretical maximum for the RP-HPLC-purified tracers and was significantly lower for the PAGE-purified tracers. Significant differences were found in the binding of different tracers to the insulin antiserum: maximum binding ranged from 94 to 99% and was significantly lower for tracers purified by RP-HPLC eluents B and C; antiserum dilution giving 50% tracer binding was lower for tracers purified by RP-HPLC eluent B. The four insulin derivatives showed no difference in non-specific precipitation and in the affinity constant values calculated from the Scatchard analysis. No significant difference was found in the binding of the four insulin derivatives to the human-cultured IM-9 lymphocytes and to the human circulating monocytes. In conclusion, the present work demonstrates that the immunological properties of the A14-[125I]monoiodoinsulin purified by RP-HPLC may be partially affected by the composition of the mobile phase. In order to obtain a fully potent A14-[125I]insulin derivative and to have the possibility of comparing data from different laboratories, the chromatographic conditions must be taken into account.