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

This laboratory, and others, have reported multiple radioactive peaks in th e size exclusion high performance liquid chromatographic (HPLC) analysis of Tc-99m-labeled peptides. In the case of one Tc-99m-MAG(3)-labeled peptide studied in this laboratory, human neutrophil elastase inhibitor, all five r adioactive peaks were shown to be due to active peptide rather than radioco ntaminants. By a variety of experiments, the nature of these peaks have now been examined. A high molecular weight UV peak could be generated by heati ng the MAG; coupled, but not the native, peptide. Furthermore, this UV peak did not appear upon heating the peptide if the sulfur within the MAG, chel ator was replaced with oxygen. This peak may therefore be due to polymers r esulting from intermolecular disulfide bond formation between sulfurs in th e MAG(3) chelate and the peptide. Several peaks with apparent lower molecul ar weights were absent on analysis with a different size exclusion column w ith 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 in teractions between the (TC)-T-99m-MAG(3) chelate and the peptide which prod uce multiple molecular configurations of identical molecular weight but dif fering in shape, charge, isomerism or lipophilicity such that they are reso lved under the conditions of certain analyses. In support of this possibili ty, lengthening the linker between MAG(3) and the peptide reduced the numbe r of radioactive peaks, while encouraging the interaction by replacing MAG( 3) with the shorter MAG(2) seemed to increase the number of radioactive pea ks. Finally, that the three "low" molecular a weight radioactive peaks reap peared when a single peak fraction was reanalyzed suggests that the species responsible ape in rapid equilibrium. One conclusion from this investigati on is that the appearance of a single peak by any HPLC analysis offers no a ssurance that multiple peaks would not appear on alternative HPLC analyses. Evidence that each species is due to radiolabeled active peptide and not t o radiocontaminants is therefore potentially more important than evidence o f a single peak. (C) 1999 Elsevier Science Ltd. All rights reserved.