Reaction of the zinc sensor FluoZin-3 with Zn(7)-metallothionein: Inquiry into the existence of a proposed weak binding site.

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. Copyright 2009 Elsevier Inc. All rights reserved.