Peroxidase (POD)

Peroxidase was determined by the procedure described by Sadasivam & Manickam (1996) with minor modifications. Guaiacol was used as substrate. The assay was performed using 50 mM phosphate buffer, a 20 mM guaiacol solution, and a 25 mM H₂O₂ solution. In a 96-well microplate (Microtiter ™), 300, 5, and 10 µL of the above solutions were placed, respectively, and, finally, 10 µL of the enzyme extract was added. The absorbance was read spectrophotometrically (xMark ™ BIO-RAD) at 436 nm. The reading of the reaction started when the reaction absorbance was 0.05 and stopped when it reached an absorbance of 0.1. The enzymatic activity was determined by the production level of tetraguaiacol. The results were expressed in U/mg protein.