The effects of calcium ion-chelating ligand (EDTA), heat, and enzyme concentration on the stability of a metalloenzyme (α-amylase, from Bacillus sources) solution have been examined by experimental as well as theoretical studies. A simple two-stage inactivation model has been presented that explicitly includes the calcium ion concentration and can explain all experimental results. The first stage involves a reversible inactivation process caused by the dissociation of a metal ion (C2+) from the active enzyme molecule, followed by the second stage of inactivation in which the apoenzyme (protein without the metal) undergoes an irreversible thermal inactivation (denaturing). These studies also suggest that the diluted enzyme solution is more prone to inactivation than the concentrated enzyme solution.
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