Purification and characterization of benzoyl-l-arginine p-nitroanilide hydrolase from etiolated leaves of Zea mays

Benzoyl-l-arginine p-nitroanilide hydrolase in the etiolated leaves of Zea mays L. has been purified 1,266-fold by a combination of gel filtration, ion exchange, and hydrophobic chromatography with a recovery of 13%. The specific activity of the purified enzyme is 5.7 units/mg protein. The enzyme is an acidic protein with a pI value of 4.6 and optimum pH of 8.2. The molecular weight of the enzyme was estimated to be 59,000. Substrate inhibition was observed at a concentration higher than 30 μM BAPA and the apparent K_m for BAPA was 29 μM at pH 8.0. The enzyme activity was inhibited by sulfhydryl reagents, leupeptin, antipain, and N-tosyl-l-lysine chloromethyl ketone. The inhibitor study suggests that the enzyme belongs to the class of the sulfhydryl proteases.