title: Reaction of metal-binding ligands with the zinc proteome: zinc sensors and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.
abstract: The commonly used Zn(2+) sensors 6-methoxy-8-p-toluenesulfonamidoquinoline (TSQ) and Zinquin have been shown to image zinc proteins as a result of the formation of sensor-zinc-protein ternary adducts not Zn(TSQ)(2) or Zn(Zinquin)(2) complexes. The powerful, cell-permeant chelating agent N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is also used in conjunction with these and other Zn(2+) sensors to validate that the observed fluorescence enhancement seen with the sensors depends on intracellular interaction with Zn(2+). We demonstrated that the kinetics of the reaction of TPEN with cells pretreated with TSQ or Zinquin was not consistent with its reaction with Zn(TSQ)(2) or Zn(Zinquin)(2). Instead, TPEN and other chelating agents extract between 25 and 35% of the Zn(2+) bound to the proteome, including zinc(2+) from zinc metallothionein, and thereby quench some, but not all, of the sensor-zinc-protein fluorescence. Another mechanism in which TPEN exchanges with TSQ or Zinquin to form TPEN-zinc-protein adducts found support in the reactions of TPEN with Zinquin-zinc-alcohol dehydrogenase. TPEN also removed one of the two Zn(2+) ions per monomer from zinc-alcohol dehydrogenase and zinc-alkaline phosphatase, consistent with its ligand substitution reactivity with the zinc proteome.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, USA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Meeusen
authors.forename: Andrew
authors.initials: A
authors.lastname: Nowakowski
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D005029:Ethylenediamines; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D020543:Proteome; D013552:Swine; D015032:Zinc / Q000737:chemistry*
chemical_list: D005029:Ethylenediamines; D008024:Ligands; D008670:Metals; D020543:Proteome; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine