title: Reaction of the zinc sensor FluoZin-3 with Zn(7)-metallothionein: Inquiry into the existence of a proposed weak binding site.
abstract: It has been reported that Zn(7)-metallothionein (MT), contains one weak binding site for Zn(2+). To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn(2+). Contrary to the previous study, no evidence was found for Zn(2+) stoichiometrically bound to the protein with an apparent stability constant of about 10(8). Indeed, stability constant measurements based upon competition between Zn(7)-MT and ligands of known stability with Zn(2+) showed that all of the protein bound Zn(2+) displayed the same stability constant at pH 7.4 and 25 degrees C of (1.7+/-0.6)x10(11). Brief reaction of Zn(7)-MT with strong acid converted it into MT(*) and upon reneutralization into Zn(7)-MT(*), which demonstrated reactivity of about 1 Zn(2+)/mol MT with competing ligands. Acid titration of Zn(7)-MT to pH 2 or below rapidly resulted in the formation of Zn(7)-MT(*) that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn(2+) between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Namdarghanbari
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Meeusen
authors.forename: Gary
authors.initials: G
authors.lastname: Bachowski
authors.forename: Nicholas
authors.initials: N
authors.lastname: Giebel
authors.forename: Jeremiah
authors.initials: J
authors.lastname: Johnson
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008668:Metallothionein / Q000378:metabolism*; D015394:Molecular Structure; D011083:Polycyclic Compounds / Q000378:metabolism*; D011817:Rabbits; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; C451182:FluoZin-3; D008024:Ligands; D011083:Polycyclic Compounds; D008668:Metallothionein; D015032:Zinc