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 Zn²⁺. 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 (λ_MAX, 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.