Catalytic Characteristics of Peroxidase from Wheat Grass

Abstract

The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk

“…The quantity of oxidised OPD was calculated from the absorbance measurement at 445 nm using an extinction coefficient (e) of 11,100 M À1 cm À1 (Lai, Wang, Chang, & Wang, 2006) according to the Lambert-Beer law. One enzyme unit was defined as the amount of enzyme required to produce one micromole of oxidised OPD per minute.…”

“…After adding 0.05 ml of 4 N HCl to stop the reaction, the absorbance at 445, 420, 295, 370 and 414 nm was measured for OPD, pyrogallol, catechol, quercetin and ABTS, respectively. The extinction coefficient values were 11,100, 2,640, 1,700, 17,500 and 31,100 M À1 cm À1 for OPD, pyrogallol, catechol, quercetin and ABTS, respectively (Lai et al, 2006;Suzuki, Honda, Mukasa, & Kim, 2006). One enzyme unit was defined as the amount of enzyme required to produce one micromole of oxidised product per minute under standard conditions.…”

“…After concentration, a 0.05-ml aliquot of the enzyme was incubated at 25°C for 30 min with 0.025 ml of reaction buffer (Lai et al, 2006) as the control or 0.025 ml of a chemical modifier. The following chemical modification reagents were tested: NBS (2 and 20 mM), EAM (1 M), NAI (10 mM), DNFB (10 mM), PMSF (10 mM), DEPC (10 mM), WRK (200 mM), and pHMB (2 mM).…”

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

“…Similarly, K M for guaiacol was reported in the range of 0.288-6.5 mM and 0.133-5.6 mM for H 2 O 2 among the peroxdiases from different sources (Lai et al 2006;Marzouki et al, 2005;Pandey and Dwivedi 2011;Suzuki et al 2006;Vernwal et al 2006). The apparent V max /K m for o-dianisidine was 449 while for H 2 O 2 it was 5×10 5 .…”

“…Peroxidase from oil palm fruit was reported to be a homotetrameric protein of 48 kDa subunit each (Deepa and Arumughan 2002). MichaelisMenten constant was reported among other peroxidases from 0.28 to 9.5 mM for guaiacol (Lai et al 2006;Marzouki et al 2005;Pandey and Dwivedi 2011;Rudrappa et al 2007;Suzuki et al 2006) and 0.133 to 5.6 mM for H 2 O 2 (Marzouki et al 2005;Pandey and Dwivedi 2011;Rudrappa et al 2007;Vernwal et al 2006). Temperature and pH optima for peroxidases from different sources such as buckwheat seed and A. sativum showed temperature optimum of 10-30°C & 35°C respectively (Suzuki et al 2006), while A. sativum, S. melongena, and B. vulgaris reported pH optimum at 5 for most of the peroxidases (Lai et al 2006;Marzouki et al 2005;Rudrappa et al 2007).…”