The ionic solutions of metals (5 mM), i.e., Cu2?, Mg2?, Mn2? and Fe2?
were prepared in acetate buffer (0.1 M, pH 6.0).
50 ll enzyme in acetate buffer (0.1 M, pH 6.0) was preincubated with 500 ll of different metal ions at 37 _C for 30 min.
After incubation, 950 ll acetate buffer followed by 500 ll sodium phytate solution (5 mM, pH 6.0)
was added to the reaction mixture.
The reaction mixture was again incubated at 37 _C for 30 min.
Reaction was stopped by adding 500 ll colour stop reagent.
The residual enzyme activity was measured at k415 nm.
The residual activity was calculated and plotted against respective metal ions.