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.