Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide

Abstract

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn2+ availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn 2+. Control cells contained proteomic Zn2+ but no detectable low molecular weight (LMW) Zn2+. With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn2+ as Zn(ZQ)2; TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)2 (λMAX, 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)2. In neither experiment was Zn2+ lost from cells. NEM altered Zn2+ accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)2 was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)2. Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn2+ sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis. © 2012 The Royal Society of Chemistry.