Reaction of Metal-Binding Ligands with the Zinc Proteome: Zinc Sensors and N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine

Abstract

The commonly used Zn2+ sensors, TSQ and Zinquin, have been shown to image Zn-proteins as a result of the formation for sensor-Zn-protein ternary adducts not Zn(TSQ)2 or Zn(Zinquin)2 complexes. The powerful, cell permeant chelating agent TPEN is also used in conjunction with these and other Zn2+ sensors to validate that the observed fluorescence enhancement seen with the sensors depends on intracellular interaction with Zn2+. We demonstrated that the kinetics of reaction of TPEN with cells pretreated with TSQ or Zinquin was not consistent with its reaction with Zn(TSQ)2 or Zn(Zinquin)2. Instead, TPEN and other chelating agents extract between 25–35% of the Zn2+ bound to the proteome, including Zn2+ from Zn-metallothionein, and, thereby, quench some but not all of the sensor-Zn-protein fluorescence. Another mechanism in which TPEN exchanges with TSQ or Zinquin to form TP EN-Zn-protein adducts found support in the reactions of TPEN with Zinquin-Zn-alcohol dehydrogenase. TPEN also removed one of the two Zn2+ ions per monomer from Zn-alcohol dehydrogenase and Zn-alkaline phosphatase, consistent with its ligand substitution reactivity with the Zn-proteome.

Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions

“…43-45 As shown elsewhere, reacting TSQ-Zn-protein adducts with TPEN results in a decrease in fluorescence through either ligand substitution or competitive adduct formation with TSQ-Zn-proteins. 29 Thus, the resulting depletion of TSQ-fluorescence after treatment with TPEN provides validity to the claim that Zn-proteins broadly maintained their metal cofactors after electrophoresis using NSDS-PAGE. Overall, these data support the conclusion that NSDS-PAGE offers a viable method for detecting protein-bound metals after electrophoresis.…”

“…Of this total, EDTA, alone, accounts for a significant fraction, about one-third, according to previous studies. 28, 29 …”

Newport Green, a fluorescent sensor of weakly bound cellular Zn2+: competition with proteome for Zn2+

“…Since TPEN forms ternary adducts with Zn-proteins, the results also suggested that NPG A had not formed NPG-Zn-protein species that could be converted to TPEN-Zn-proteins. 25 …”

The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn2+ -chelation and inhibiting the serine protease Omi/HtrA2

“…Zinquin fluorescence was punctate consistent with sequestration into endosomes, lysososmes, or other organelles [29] while FluoZin fluorescence was diffuse consistent with predominantly cytoplasmic distribution. While the origin of Zn 2+ -dependent fluorescence by Zinquin and FluoZin remains under investigation and apparently involves interaction of the fluorphores with Zn 2+ -binding proteins rather than with an aqueous free-Zn 2+ pool in cells [30], the results are consistent with ZnPy cytotoxicity being mediated through increased intra-cellular Zn 2+ levels.…”