Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site

It has been reported that Zn7-metallothionein (MT), contains one weak binding site for Zn2+. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn2+. Contrary to the previous study, no evidence was found for Zn2+ stoichiometrically bound to the protein with an apparent stability constant of about 108. Indeed, stability constant measurements based upon competition between Zn7-MT and ligands of known stability with Zn2+ showed that all of the protein bound Zn2+ displayed the same stability constant at pH 7.4 and 25 °C of (1.7 ± 0.6) × 1011. Brief reaction of Zn7-MT with strong acid converted it into MT* and upon reneutralization into Zn7-MT*, which demonstrated reactivity of about 1 Zn2+/mol MT with competing ligands. Acid titration of Zn7-MT to pH 2 or below rapidly resulted in the formation of Zn7-MT* that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn2+ 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.