Detection of Zn2+ release in nitric oxide treated cells and proteome: dependence on fluorescent sensor and proteomic sulfhydryl groups

Abstract

Nitric oxide (NO) is both an important regulatory molecule in biological systems and a toxic xenobiotic. Its oxidation products react with sulfhydryl groups and either nitrosylate or oxidize them. The aerobic reaction of NO supplied by diethylamine NONOate (DEA-NO) with pig kidney LLC-PK₁ cells and Zn-proteins within the isolated proteome was examined with three fluorescent zinc sensors, zinquin (ZQ), TSQ, and FluoZin-3 (FZ-3). Observations of Zn²⁺ labilization from Zn-proteins depended on the specific sensor used. Upon cellular exposure to DEA-NO, ZQ sequestered about 13% of the proteomic Zn²⁺ as Zn(ZQ)₂ and additional Zn²⁺ as proteome·Zn–ZQ ternary complexes. TSQ, a sensor structurally related to ZQ with lower affinity for Zn²⁺, did not form Zn(TSQ)₂. Instead, Zn²⁺ mobilized by DEA-NO was exclusively bound as proteome·Zn–TSQ adducts. Analogous reactions of proteome with ZQ or TSQ in vitro displayed qualitatively similar products. Titration of native proteome with Zn²⁺ in the presence of ZQ resulted in the sole formation of proteome·Zn–ZQ species. This result suggested that sulfhydryl groups are involved in non-specific proteomic binding of mobile Zn²⁺ and that the appearance of Zn(ZQ)₂ after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ)₂ instead of proteome·Zn–ZQ. With the third sensor, FluoZin-3, neither Zn–FZ-3 nor proteome·Zn–FZ-3 was detected during the reaction of proteome with DEA-NO. Instead, it reacted independently with DEA-NO with a modest enhancement of fluorescence.