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Ferroxidase assay of Haliangium ochraceum encapsulin encapsulin:encapsulated ferritin complexes

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posted on 29.09.2021, 08:46 by Jon Marles-WrightJon Marles-Wright

The enzymatic activity of Empty-Enc and Loaded-Enc were assessed by ferroxidase assay. Fe(II) samples were prepared by dissolving FeSO4.7H2O in HCl 0.1 % (v/v) under anaerobic conditions. Protein samples were diluted anaerobically in Buffer GF (20 mM HEPES, pH 8.0, 150 mM NaCl) to a final encapsulin monomer concentration of 9 µM to allow comparison between experiments.

Iron and protein aliquots were added aerobically to a quartz cuvette (Hellma) resulting in a final concentration of 100 μM iron and 15 μM (Loaded-Enc), or 9 μM (Empty-Enc). The cuvette was placed in a UV-visible spectrophotometer (PerkinElmer Lambda 35) and the reaction sample was incubated at 21°C for 50 s to stabilise. Absorbance at 315 nm was then recorded every second for 1450 s using the Time-Drive software. A control experiment was conducted by monitoring the background oxidation by atmospheric oxygen of 100 μM FeSO4*7H2O in the absence of the enzyme.

Loaded-Enc experiments were carried out with three biological replicates. There were six technical replicates for batch one, two relplicates using batch two and one replicate from batch three. Means and standard deviations were calculated on the time zero-subtracted progress curves.


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