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Merck

Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts.

Bio-protocol (2017-09-20)
Mareike Hauenstein, Stefan Hรถrtensteiner
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Hydroxylation of chlorophyll catabolites at the so-called C32 position ( Hauenstein et al., 2016 ) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the substrate epi-pFCC (epi-primary Fluorescent Chlorophyll Catabolite) ( Mรผhlecker et al., 2000 ) to epi-pFCC-OH.

MATERIALS
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Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Ferredoxin from Spinacia oleracea (spinach), lyophilized powder, Ferredoxin โ‰ฅ15 wt. %
Sigma-Aldrich
Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides, recombinant, expressed in E. coli, ammonium sulfate suspension, โ‰ฅ550 units/mg protein (biuret)
Sigma-Aldrich
Ferredoxin-NADP+ Reductase from Spinacia oleracea (spinach), lyophilized powder, โ‰ฅ15 units/mg solid, secondary activity: โ‰ฅ10 units/mg solid NADPH diaphorase