Purification and characterization of a peroxidase from Calotropis procera latex and its distinct role from anthelmintic proteins

This study reports the proteomic analysis of C. procera latex, as well as the identification, purification, and partial characterization of an unusual peroxidase (CpLP-POX). Initially, the proteomic analysis revealed typical defense-related proteins, such as proteases, chitinases, and osmotins, along with a high-molecular-mass peroxidase. Sequential ion-exchange (CM-Sepharose) and gel filtration chromatography (Sephacryl S-100) yielded a homogeneous peroxidase (CpLP-POX) whose native form dissociated into ∼35 kDa subunits under denaturing conditions, suggesting a trimeric organization in its native state. Molecular modeling showed that CpLP-POX monomers exhibit a conserved catalytic site, four disulfide bonds, and an overall folding typical of class III plant peroxidases. CpLP-POX displayed maximal activity at pH 6.0 and 40 °C and was inhibited by EDTA (98% at 1 mM), sodium azide (33% at 10 mM), and thiol-reducing agents such as DTT (70% at 0.5 mM), consistent with a Ca²⁺-dependent heme peroxidases. Kinetic analysis revealed classical Michaelis-Menten behavior, with apparent Km values of 14.3 mM for guaiacol and 2.8 mM for hydrogen peroxide. CpLP-POX showed no inhibitory effect on nematode egg hatching even at 10 mg/mL, indicating that it does not contribute directly to the anthelmintic activity. Altogether, this study provides a comprehensive initial biochemical framework for a new latex peroxidase, expanding current knowledge of plant peroxidases while highlighting the need for further high-resolution structural and mechanistic investigations.