Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at le ast three cysteine proteases with high proteolytic activity, called ervatam ins. One of these proteases, named ervatamin B, has been purified to homoge neity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtra tion. The extinction coefficient (epsilon(280)(1%) (nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hy drolyzed denatured natural substrates such as casein, azoalbumin, and azoca sein with a high specific activity. In addition, it showed amidolytic activ ity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an appare nt K-m and K-cat of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-1), respectively. Th e pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalb umin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was str ongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine protease s of the papain superfamily. The enzyme was relatively stable toward pH, de naturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's doub le immunodiffusion and typical color in ELISA. Other related proteases do n ot cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino ac id residues and considerable similarity to typical plant cysteine proteases .