No 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.

The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.