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 microM BAPA and the apparent Km for BAPA was 29 microM 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.