Petunia peroxidase a: isolation, purification and characteristics.
The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.
Hendriks
T
T
Department of Plant Physiology, Agricultural University, Wageningen, The Netherlands.
Wijsman
H J
HJ
van Loon
L C
LC
D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006139:Guaiacol / Q000037:antagonists & inhibitors / Q000737:chemistry; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D010545:Peroxides / Q000737:chemistry; D010636:Phenols / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity
D007527:Isoenzymes; D010545:Peroxides; D010636:Phenols; D006139:Guaiacol; C010559:coniferyl alcohol; D010544:Peroxidases