Barley leaf peroxidase: purification and characterization.
Peroxidase was prepared from extracts of barley leaves and separated into seven components, different in pI. The purification procedure comprised two parts. The first part was based on the fact that all the components had practically the same molecular weights. It consisted of fractionations with acetone and ammonium sulfate, ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose CL-6B, and molecular-sieve chromatography on Ultrogel AcA44; the components were all purified together to near homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the procedure resulted in 1,200-fold purification with a yield of 39%. The ion-exchange chromatographies were carried out under conditions such that the components would not be adsorbed. In the second part, the enzyme preparation was separated into the seven components by repeating isoelectric electrophoresis. Their isoelectric points (pI) were 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. The components other than the pI 6.3 and 6.8 components were each purified to homogeneity in the electrophoresis. The seven components thus prepared were the same in molecular weight on SDS-gel electrophoresis (44,000) and showed absorption maxima at the same wave-lengths (403, 496, and 534 nm), RZ (A403/A275) ranging from 2.09 to 2.81. Their protoheme IX contents were 0.81-1.07 mol/mol, and their true sugar contents 15-26% (g/g). The amino acid compositions suggest that the five components described above are not real isoenzymes, but exhibit different pI values due to differences in glycosyl residue. The pI 9.3 component was crystallized in spite of its high sugar content.
D003460:Crystallization; D001467:Hordeum / Q000201:enzymology; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification*; D008970:Molecular Weight; D009195:Peroxidase; D010544:Peroxidases / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D013053:Spectrophotometry
D007527:Isoenzymes; D010544:Peroxidases; D009195:Peroxidase