Catalytic characteristics of peroxidase from wheat grass.
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).
Lai
Lih-Shiuh
LS
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan 402, Republic of China. lslai@dragon.nchu.edu.tw
Wang
Dai-Jung
DJ
Chang
Chen-Tien
CT
Wang
Cheng-Hsin
CH
D002021:Buffers; D002384:Catalysis; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D010544:Peroxidases / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity
D002021:Buffers; D007477:Ions; D008670:Metals; D012492:Salts; D006859:Hydrogen; D010544:Peroxidases; C070865:wheat peroxidase