Purification and Characterization of a Stable Cysteine Protease Ervatamin B, with Two Disulfide Bridges, from the Latex ofErvatamiacoronaria

Abstract

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin 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 strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.

Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

“…Ervatamin B was purified as described (Kundu et al, 2000). Spectroscopic grade methanol, ethanol, 2-propanol, TFE, acetonitrile and Molecular Biology grade glycerol were purchased from the Sigma Chemical Co. (St. Louis, USA).…”

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

“…Materials Fresh latex, obtained from young stems of the plant Ervatamia coronaria, was used to purify the enzyme ervatamin B, as described previously (Kundu et al, 2000). Sodium tetrathionate was used throughout the purification procedure to avoid any complications due to autodigestion, and this inactive enzyme was used for all of the biophysical studies reported here.…”

“…All of the physical properties of the enzyme are the same in both the active and inactive forms. Concentration of the enzyme was determined by spectrophotometry using an extinction coefficient ε 1cm 1% = 20.5 at 280 nm (Kundu et al, 2000) and also by Bradford assay. All other materials were as described previously .…”

“…Assay for enzyme activity The hydrolyzing activity of the enzyme (under various conditions of pH and in the presence of chemical denaturant) was monitored using the denatured natural substrate azoalbumin, following the described procedure (Kundu et al, 2000). A control assay was performed with no enzyme in the reaction mixture and used as a reference.…”

“…With this view, conformational studies in solution have been initiated with ervatamin B, a novel plant cysteine protease that was isolated in our laboratory from the latex of a medicinal plant Ervatamia coronaria (Kundu et al, 2000). Ervatamin B seems to be a potential endopeptidase with many interesting features and probable applications.…”