Imidazoles as well as thiolates in proteins bind technetium-99m

99mTc is widely thought to directly bind proteins through thiolate groups of cysteine residues, resulting in Tc-cysteinyl-protein bonds. Chemical reduction of disulfide bonds in proteins is widely used to generate thiolates with the goal of increasing 99mTc binding. This strategy is used because most proteins contain no thiolates, but many do contain disulfide bonds. In this study, we have evaluated the hypothesis that imidazole groups of histidine are also involved in direct 99mTc binding to proteins. Human gamma-globulin was used as the model protein in these studies. The immunoglobulin was used (a) without reduction or was (b) treated with stannous ions to reduce disulfide bonds thereby increasing thiolate concentration. These proteins were used to evaluate the hypothesis that imidazole as well as thiolate groups bind Tc. The proteins were evaluated by (a) using free amino acids to compete with proteins for 99mTc and (b) by chemical modification of amino acid side chains. In addition, peptides known to contain either cysteine or histidine, but not both, were also successfully directly labeled with 99mTc. These results indicate that in proteins (and peptides) imidazole-containing groups as well as thiolate-containing groups bind Tc.