The metal ions; cobalt (Co²⁺), magnesium (Mg²⁺), zinc (Zn²⁺), nickel (Ni²⁺) and copper (Cu²⁺) were used at 2 mM. Four different protease inhibitors were also tested at different final concentrations, 50 μM chymostatin (inhibits chymotrypsin-like proteases), 10 μM E-64 (highly selective cysteine protease inhibitor), 100 μM leupeptin (inhibitor of serine and thiol proteases) and 1 mM phenylmethanesulfonyl fluoride (PMSF; a common serine protease inhibitor). The enzyme reaction was performed as mentioned above with all three synthetic fluorescent substrates. Percent remaining activity was calculated using control activity in the absence of inhibitor and the enzyme activity obtained in the present of inhibitor. The data are represented as mean ± S.D. for at least three independent experiments.