Reversible inactivation of rabbit muscle aldolase by o-phenanthroline

Abstract

Rabbit muscle aldolase is progressively inactivated when incubated in the presence of low concentrations of o-phenanthroline. This inactivation is caused by the oxidation of sulfhydryl groups to disulfides by atmospheric oxygen, catalyzed by a metal- o-phenanthroline complex. It is prevented by the presence of metals which bind o-phenanthroline, as well as by chelating reagents which react with traces of copper. The inactivated enzyme is fully reactivated by reduction with mercaptoethanol and partially reactivated by treatment with cyanide or borohydride, but is not reactivated by any of a large number of metal ions tested. Inactivation of the enzyme is associated with the formation of 4–5 disulfide bonds, all of which appear to be intrasubunit in nature. Inactivation in the presence of cupric- o-phenanthroline is prevented by the presence of substrates and substrate analogues. These and other results suggest that the enzyme contains 4–5 essential sulfhydryl groups, or approximately one or two per active center, which are capable of forming disulfide bridges involving an equal number of nonessential sulfhydryl groups.