Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins
Abstract
This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.
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Neutralization with sodium hydroxide shifts the absorbance of protein-conjugated hydrazones from 370 nm to 450 nm, thereby eliminating interference with unbound DNPH.
“Neutralization shifts the absorbance of protein-conjugated hydrazones from 370 nm to 450 nm, thereby eliminating the interference with unbound DNPH.”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [83]
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Shields, H. J., Traa, A., & Van Raamsdonk, J. M. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.628157 · Cited by 478 · see citing works
Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases
Kehm, R., Baldensperger, T., Raupbach, J., & Höhn, A. (2021) · Redox Biology · doi:10.1016/j.redox.2021.101901 · Cited by 348 · see citing works
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- Authors
- Mesquita, C. S., Oliveira, R., Bento, F., Geraldo, D., Rodrigues, J. V., & Marcos, J. C.
- Affiliations
- University of Minho (PT)
- Corresponding author
- Marcos, João C.
- Venue
- Analytical Biochemistry
- Publisher
- Elsevier BV
- Published
- 2014-08
- Volume
- 458
- Pages
- 69-71
- ISSN
- 0003-2697 (ISSN-L)
- OpenAlex
- W1993566791
- Cited by
- 480
- References
- 0
- Citations by year
- 2026: 60 · 2025: 62 · 2024: 61 · 2023: 55 · 2022: 71 · 2021: 54 · 2020: 32 · 2019: 35 · 2018: 27 · 2017: 9 · 2016: 9 · 2015: 4 · 2014: 1
- FWCI
- 3.15
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Advanced Glycation End Products research
- Keywords
- 2,4-dinitrophenylhydrazine, carbonyl compounds, oxidized proteins, protein oxidation, spectrophotometric assay, sodium hydroxide treatment, interference suppression, method comparison
- MeSH terms
- Oxidation-Reduction, Phenylhydrazines, Proteins, Sodium Hydroxide, Spectrophotometry
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