Zn2+ is a necessary cofactor for thousands of mammalian proteins. Research has suggested that transient fluxes of cellular Zn2+ are also involved in processes such as apoptosis. Observations of Zn2+ trafficking have been collected using Zn2+ responsive fluorescent dyes. A commonly used Zn2+ fluorophore is TSQ. The chemical species responsible for TSQ's observed fluorescence in resting or activated cells have not been characterized. Parallel fluorescence microscopy and spectrofluorometry of LLC-PK1 cells incubated with TSQ demonstrated punctate staining that concentrated around the nucleus and was characterized by an emission maximum near 470 nm. Addition of cell permeable Zn-pyrithione resulted in greatly increased, diffuse fluorescence that shifted the emission peak to 490 nm, indicative of the formation of Zn(TSQ)2. TPEN, a cell permeant Zn2+ chelator, largely quenched TSQ fluorescence returning the residual fluorescence to the 470 nm emission maximum. Gel filtration chromatography of cell supernatant from LLC-PK1 cells treated with TSQ revealed that TSQ fluorescence (470 nm emission) eluted with the proteome fractions. Similarly, addition of TSQ to proteome prior to chromatography resulted in 470 nm fluorescence emission that was not observed in smaller molecular weight fractions. It is hypothesized that Zn-TSQ fluorescence, blue-shifted from the 490 nm emission maximum of Zn(TSQ)2, results from ternary complex, TSQ-Zn-protein formation. As an example, Zn-carbonic anhydrase formed a ternary adduct with TSQ characterized by a fluorescence emission maximum of 470 nm and a dissociation constant of 1.55 × 10-7 M. Quantification of TSQ-Zn-proteome fluorescence indicated that approximately 8% of cellular Zn2+ was imaged by TSQ. These results were generalized to other cell types and model Zn-proteins.

Zinquin (ZQ) is a commonly used sensor for cellular Zn2+ status. It has been assumed that measures accessible Zn2+ concentrations in the nmolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn2+ bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)2 with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)2, it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn2+. Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH) and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQACID able to bind to one Zn2+ and extract the other in Zn2-ADH, whereas it removed one Zn2+ from Zn2-AP and did not bind to the other. ZQETHYL ESTER bound to both proteins without sequestering Zn2+ from either one. In contrast to a closely related sensor, TSQ, neither ZQACID nor ZQEE associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn2+ bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn2+ proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.

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.

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.