title: Procerain, a stable cysteine protease from the latex of Calotropis procera.
abstract: A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
authors.forename: Vikash Kumar
authors.initials: VK
authors.lastname: Dubey
authors.forename: M V
authors.initials: MV
authors.lastname: Jagannadham
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D031107:Calotropis / Q000201:enzymology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D005779:Immunodiffusion / Q000379:methods; D007526:Isoelectric Point; D007840:Latex / Q000737:chemistry*; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000596:Amino Acids; D015853:Cysteine Proteinase Inhibitors; D007840:Latex; D003546:Cysteine Endopeptidases; C474045:procerain