title: Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution.
abstract: Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) 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 Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) 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.
authors: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, USA.
authors: Andrew B
authors: AB
authors: Nowakowski
authors: David H
authors: DH
authors: Petering
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000634:Aminoquinolines / Q000737:chemistry; D045744:Cell Line, Tumor; D004952:Esters; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D015363:Quinolones / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014105:Tosyl Compounds / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D004952:Esters; D005456:Fluorescent Dyes; D008024:Ligands; D020543:Proteome; D015363:Quinolones; D014105:Tosyl Compounds; C061730:N-(6-methoxy-8-quinolyl)-4-toluenesulfonamide; D000426:Alcohol Dehydrogenase; D015032:Zinc; C084731:zinquin