Zinc trafficking: 1,10-phenanthroline, glutathione, and other metal binding ligands form adducts with proteomic Zn2+

Hypotheses were tested that the proteome of pig kidney LLC-PK1 cells (i) contains Zn-proteins that react with a diversity of native and pharmacologically active metal-binding ligands to form ternary complexes and (ii) includes proteins that bind Zn2+ nonspecifically and together form ternary adducts with a variety of metal-binding agents. The method to observe ternary complex formation with Zn-proteins and proteome center dot Zn involved preformation of fluorescent TSQ [6-Methoxy-(8-p-toluenesulfonamido)quinoline]-Zn-proteins and/or proteome center dot Zn-TSQ adducts followed by competitive reaction with selected ligands. The loss of TSQ-dependent fluorescence signaled the replacement of TSQ by the competing ligand in the starting adducts. In vitro, 1,10-phenanthroline competed effectively with TSQ for binding to Zn-proteins in the proteome. The successful competition of 1,10-phenanthroline with TSQ-Zn-proteins was also observed in cells. Similarly, 1,10-phenanthroline was shown to bind to a sizable fraction of Zn2+ associated adventitiously with proteome (proteome center dot Zn). Other synthetic ligands that bind to Zn-proteins and proteome center dot Zn include 2,2-bipyridyl, 8-hydroxyquinoline, 2,2MODIFIER LETTER PRIME-dicarboxypyridine, and pyrithione. Such results suggest that ligand binding to such sites may play a role in the observed biological effects of these and other metal-binding molecules. Although cysteine does not significantly compete with TSQ, glutathione displaces TSQ from Zn-proteins and proteome center dot Zn at concentrations well below those found in cells, implying that ternary complex formation involving glutathione may be physiologically significant.