Abstract
An Escherichia coli expression system has been developed for pea cytosolic ascorbate peroxidase (APX). The enzyme was expressed as a fusion product with the E. coli maltose-binding protein for rapid, affinity chromatography purification. Recombinant ascorbate peroxidase (rAPX) was purified by tryptic digestion to separate the maltose-binding protein from rAPX followed by three chromatographic steps. The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots. Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center. rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected.
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Claims 6
Claim · model-written summary
Recombinant rAPX showed electrophoretic, enzymatic and spectral properties identical to native APX from pea shoots.
- Taxon
- pea
Lathyrus oleraceuse0 - Material
- rAPXe1
- Outcome
- electrophoretic, enzymatic, and spectral propertiese1
- Direction
- identicale1
- Comparator
- native APX isolated from pea shootse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e1“The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots”
Claim · model-written summary
Adding an equal molar amount of H2O2 to rAPX gave an initial decrease in the Soret maximum.
- Taxon
- peaLathyrus oleraceuse0
- Treatment
- an equal molar amount of H2O2e2
- Outcome
- Soret maximume2
- Direction
- initial decreasee2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e2“Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center”
Claim · model-written summary
The rAPX Soret peak stabilised as a red-shifted peak similar to cytochrome c peroxidase Compound I.
- Taxon
- peaLathyrus oleraceuse0
- Outcome
- stable, red-shifted Soret peake2
- Direction
- red-shiftede2
- Comparator
- cytochrome c peroxidase Compound Ie2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e2“Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center”
Claim · model-written summary
rAPX Compound I appears to consist of an oxyferryl (Fe(4+)-O) center.
- Taxon
- peaLathyrus oleraceuse0
- Outcome
- Compound Ie2
- Qualifier
- oxyferryl (Fe(4+)-O) centere2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e2“Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center”
Claim · model-written summary
A preliminary set of native diffraction data to 2.6 A was collected from rAPX crystals.
- Taxon
- peaLathyrus oleraceuse0
- Material
- rAPXe3
- Outcome
- a preliminary set of native datae3
- Value
- 2.6 Ae3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e3“rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected”
Claim · model-written summary
rAPX was crystallised in a form suitable for crystal structure determination.
- Taxon
- peaLathyrus oleraceuse0
- Outcome
- crystallizede3
- Qualifier
- in a form suitable for crystal structure determinatione3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pea cytosolic ascorbate peroxidase”
e3“rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected”