title: 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-PK1cells 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)2and 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)2after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ)2instead 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. petering@uwm.edu.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Karim
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D006834:Hydrazines / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008667:Metalloproteins / Q000032:analysis / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism*; D011083:Polycyclic Compounds / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015363:Quinolones / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism; D013552:Swine; D014105:Tosyl Compounds / Q000032:analysis / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
chemical_list: C451182:FluoZin-3; D005456:Fluorescent Dyes; D006834:Hydrazines; D008667:Metalloproteins; D020030:Nitric Oxide Donors; D011083:Polycyclic Compounds; D020543:Proteome; D015363:Quinolones; D013438:Sulfhydryl Compounds; D014105:Tosyl Compounds; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D015032:Zinc; C084731:zinquin