Purification of an infection-related acidic peroxidase from pearl millet seedlings

Abstract

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE-Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS-PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27-60°C). The K_m for H₂O₂ and V_max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl₂ dependent and both MgCl₂ and ZnCl₂ showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.