Related Experiment Video
Updated: Oct 18, 2025
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Accurate and Precise Protocol to Estimate the Activity of Peroxiredoxin Enzyme
Thulfeqar Hamza1,2, Mahmoud Hussein Hadwan2
1Chemistry Dept., College of Science, University of Babylon, Iraq.
Background:
Accurate estimation of Prx activity poses many complications and interferences. The present protocol is free of interference and provides an effective alternative for the assessment of peroxide with high sensitivity. The assay can be used in clinical pathology laboratories since it is simple, rapid, and inexpensive. The systematic reagent consisted of AFS/ASA which acted as a sensitive probe for peroxide.
Methods:
Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA).
Results:
Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of strong acid to inhibit the Prx reaction.
Conclusion:
We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method.
