Technetium-99m labeled peptides--an investigation of multiple HPLC peaks

This laboratory, and others, have reported multiple radioactive peaks in the size exclusion high performance liquid chromatographic (HPLC) analysis of 99mTc-labeled peptides. In the case of one 99mTc-MAG3-labeled peptide studied in this laboratory, human neutrophil elastase inhibitor, all five radioactive peaks were shown to be due to active peptide rather than radiocontaminants. By a variety of experiments, the nature of these peaks have now been examined. A high molecular weight UV peak could be generated by heating the MAG3 coupled, but not the native, peptide. Furthermore, this UV peak did not appear upon heating the peptide if the sulfur within the MAG3 chelator was replaced with oxygen. This peak may therefore be due to polymers resulting from intermolecular disulfide bond formation between sulfurs in the MAG3 chelate and the peptide. Several peaks with apparent lower molecular weights were absent on analysis with a different size exclusion column with superior resolution in their molecular weight range. More importantly, they were also absent on analysis by SDS polyacrylamide gel electrophoresis. These "low" molecular weight radioactive peaks may therefore be due to interactions between the 99mTc-MAG3 chelate and the peptide which produce multiple molecular configurations of identical molecular weight but differing in shape, charge, isomerism or lipophilicity such that they are resolved under the conditions of certain analyses. In support of this possibility, lengthening the linker between MAG3 and the peptide reduced the number of radioactive peaks, while encouraging the interaction by replacing MAG3 with the shorter MAG2 seemed to increase the number of radioactive peaks. Finally, that the three "low" molecular weight radioactive peaks reappeared when a single peak fraction was reanalyzed suggests that the species responsible are in rapid equilibrium. One conclusion from this investigation is that the appearance of a single peak by any HPLC analysis offers no assurance that multiple peaks would not appear on alternative HPLC analyses. Evidence that each species is due to radiolabeled active peptide and not to radiocontaminants is therefore potentially more important than evidence of a single peak.