| Budget Amount *help |
¥21,800,000 (Direct Cost: ¥21,800,000)
Fiscal Year 1985: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1984: ¥20,000,000 (Direct Cost: ¥20,000,000)
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| Research Abstract |
Peroxidase (EC 1.11.1.7) is a protein bound with heme as the prosthetic group, which catalyses the oxidation of appropriate electron donors by hydrogen peroxide. This enzyme has been studied biochemically for a long time, because of its wide distribution among living organisms, and strong catalytic activity with various reagents. On the other hand, little structural study on peroxidase has been reported. This project has been carried out to extract and purify the barley leaf peroxidase , to crystallize the enzyme, and to undertake X-ray diffraction experiment on the crystal.
Peroxidase was purified from extracts of barley leaves and separated into seven isozymes with different pI's, 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. All the isozymes have the same molecular weight of 44,000 dalton including one protoheme and about 20% of oligosaccharides. The crystallization of isoenzymes with pI's 7.4 and 9.3 have been tried, which are relatively easy to isolate from the other isozymes and have high yields and specific activities. Among the various crystallization conditions examined, the microdialysis with polyethylene glycol 4,000 as precipitant gave brown plate crystals of isozyme with pI 9.3. The unit cell dimensions are determined ca. 52, 55, and 141 <゜]A> . When the four peroxidase molecules are included in the unit cell, the unit cell volume per unit molecular weight, <V_m> , is calculated as 2.4 <゜]A^3> /dalton, which is comparable with the reported values of 1.68-3.53 <゜]A^3> /dalton. Further X-ray experiments are in progress.
The purifications and crystallizations of superoxide dismutase and ferredoxin <NADP^+> oxidoreductase from the extracts of barley leaves have also been carried out.
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