Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h I) was purified to mass spectroscopy homogeneit y from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultr acentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9. 3. The optimum pH range for casein hydrolysis was 8.0-9.5. The enzyme showe d remarkable caseinolytic activity at high temperatures, although its therm al stability decayed rapidly. The proteinase was activated by thiol compoun ds and inhibited by common thiol-blocking reagents, particularly E-64 and H gCl2, suggesting the enzyme belongs to the cysteine protease family. The co ncentration of active sites as determined by titration with E-64 was 3.3 mu M. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl eaters, the enzyme showed higher preference for the glutamine derivative, followed by those o f alanine, asparagine, glycine, and leucine, in decreasing order. Partial h omology (36-48%) with other plant cysteine proteinases was observed in an i nternal fragment obtained by Protease Vs treatment.