Novel Hydrogen Peroxide Metabolism in Suspension Cells ofScutellaria baicalensis Georgi

Abstract

We identified a rapid and novel system to effectively metabolize a large amount of H 2 O 2 in the suspension cells of Scutellaria baicalensis Georgi. In response to an elicitor, the cells immediately initiate the hydrolysis of baicalein 7-O-␤-D-glucuronide by ␤-glucuronidase, and the released baicalein is then quickly oxidized to 6,7-dehydrobaicalein by peroxidases. Hydrogen peroxide is effectively consumed during the peroxidase reaction. The ␤-glucuronidase inhibitor, saccharic acid 1,4-lactone, significantly reduced the H 2 O 2 -metabolizing ability of the Scutellaria cells, indicating that ␤-glucuronidase, which does not catalyze the H 2 O 2 degradation, plays an important role in the H 2 O 2 metabolism. As H 2 O 2 -metabolizing enzymes, we purified two peroxidases using ammonium sulfate precipitation followed by sequential chromatography on CM-cellulose and hydroxylapatite. Both peroxidases show high H 2 O 2 -metabolizing activity using baicalein, whereas other endogenous flavones are not substrates of the peroxidase reaction. Therefore, baicalein predominantly contributed to H 2 O 2 metabolism. Because ␤-glucuronidase, cell wall peroxidases, and baicalein pre-exist in Scutellaria cells, their constitutive presence enables the cells to rapidly induce the H 2 O 2 -metabolizing system.

Identification and Molecular Characterization of Novel Peroxidase with Structural Protein-like Properties

“…Purification of Peroxidases-The fraction containing cationic peroxidases was prepared from 4-week-old callus (300 g) by ammonium sulfate precipitation, followed by chromatography on CM-cellulose (19). Subsequently, this fraction was applied to a hydroxylapatite column (Nacalei Tesque, Kyoto, Japan, 1.0 ϫ 20.0 cm) preequilibrated with 10 mM phosphate buffer (pH 7.0).…”

“…However, the motifs characteristic of extensins and PRPs were not found. Previously, we identified two cationic peroxidase isozymes (peroxidases 1 and 2) catalyzing H 2 O 2 -metabolism in S. baicalensis (19), and the molecular mass of the 36-kDa protein was closer to peroxidase 1 than peroxidase 2. Finally, the 36-kDa protein was identified as peroxidase 1 by Western blotting using affinity-purified anti-peroxidase 1 antibody (Fig.…”

“…The insolubilization of extensins and PRPs, which is initiated within 2-5 min and completed within 20 -30 min, is one of the earliest defense responses (1), whereas the insolubilization of peroxidase 1 was somewhat slower. We hypothesized that this lower rate is due to scarcity of H 2 O 2 , because Scutellaria cells rapidly metabolize large amounts of H 2 O 2 produced during oxidative burst (19). Hence, we conducted experiments using an excess of H 2 O 2 , but only a slight increase in the insolubilization rate occurred (Fig.…”

“…Previously, we identified a novel H 2 O 2 -detoxifying system induced by elicitor treatment in Scutellaria cells and demonstrated that two ionically bound wall peroxidases (peroxidases 1 and 2) effectively metabolize large amounts of H 2 O 2 produced during oxidative burst (19). Further studies have revealed that peroxidase 1 possesses quite novel functions; like extensins and PRPs, peroxidase 1 is covalently bound to cell wall in response to elicitor treatment and mechanical damage, and surprisingly, this reaction is catalyzed by the action of peroxidase 1 itself in the presence of H 2 O 2 .…”

Purification and characterization of baicalin-β-d-glucuronidase hydrolyzing baicalin to baicalein from fresh roots of Scutellaria viscidula Bge

“…All this proves that it is easy to transform baicalin into baicalein. It is noticed that there is cell wall peroxidases in skullcaps, which results in baicalein oxidation to 6,7-dehydrobaicalein [20] and accordingly weaken the pharmacological effect. Therefore, it is necessary to insure peroxidases denaturation in transforming baicalin into baicalein.…”