Reaction of the Zinc Sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the Existence of a Proposed Weak Binding Site

摘要

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⁺ stoichiometically 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.