title: Biochemical and spectroscopic characterization of a novel metalloprotease, cotinifolin from an antiviral plant shrub: Euphorbia cotinifolia.
abstract: A high molecular mass novel metalloprotease, cotinifolin is purified from the latex of Euphorbia cotinifolia by a combination of anion exchange and hydrophobic interaction chromatography. The nonglycosylated enzyme has a molecular mass of 79.76 kDa (ESI-MS) and the isoelectric point of the enzyme is pH 7.7. Cotinifolin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity. The K(m) value of the enzyme was found to be 20 μM with azocasein. The enzyme is not prone to autolysis even at very low concentrations. Polyclonal antibodies specific to enzyme was raised and immunodiffusion reveals that the enzyme has unique antigenic determinants. Maximum caseinolytic activity of cotinifolin is observed in the range of pH 7.0-8.0 and temperature of 50 °C. Using 0.2 mL of 1 mM solution of each metal ion, the purified protease was inhibited slightly by Ba²⁺ and Mn²⁺, moderately by Mg²⁺, Ca²⁺ and Cs²⁺ and significantly by Zn²⁺, Cu²⁺ and Co²⁺. On the other hand, substantial activation in caseinolytic activity was achieved by Ni²⁺. The enzyme activity was also inhibited by EDTA and o-phenanthroline but not by any other protease inhibitors. Perturbation studies by temperature, pH, and chaotrophs of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Spectroscopic studies reveal that cotinifolin has secondary structural features with α/β type with approximately 9% of α-helicity. Easy availability and simple purification procedure makes the enzyme a good system for biophysical study, biotechnological and industrial applications.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
authors.forename: Reetesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Kunwar Awaneesh
authors.initials: KA
authors.lastname: Singh
authors.forename: Ritu
authors.initials: R
authors.lastname: Tomar
authors.forename: Medicherla V
authors.initials: MV
authors.lastname: Jagannadham
mesh_terms: D002413:Cations, Divalent / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D028481:Euphorbia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010946:Plants, Medicinal / Q000201:enzymology*; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D003902:Detergents; D010940:Plant Proteins; D045726:Metalloproteases