An assessment of 2,4-dinitrophenylhydrazine (DNPH)-detectable protein-based carbonyls is one ofthe most common assays used to quantify oxidative stress
in vitro and
in vivo. In this study, we compared,for the lipid-binding protein
![](/images/gifchars/beta2.gif)
-lactoglobulin, the extent to which carbonyl reactivity could be introducedby adventitious metal-catalyzed oxidation (MCO) in the absence and presence of a polyunsaturated lipidor by treatment with various individual bifunctional lipid oxidation products capable of introducingcarbonyls into proteins by adduction to nucleophilic side chains. With metal ions and either O
2/r
eductantor H
2O
2 as the terminal oxidant, the maximal level of DNPH-detectable carbonyl generation obtainablein several hours was 0.1-0.2 mol carbonyl per mol protein monomer, with Cu(II) being more effectivethan Fe(II). Exposure instead to bifunctional lipoxidation-derived aldehydes (1-2 mM) generated insome cases in excess of 1 mol carbonyl per mol protein. The rank order of carbonyl incorporation reactivitywas acrolein > 4-oxo-2-nonenal > 4-hydroxy-2-nonenal > 2,4-decadienal > malondialdehyde. Proteincross-linking ability followed a somewhat different rank order. Parallel studies on r
eductively methylated
![](/images/gifchars/beta2.gif)
-lactoglobulin revealed that His and Cys residues are intrinsically more responsible than Lys residuesfor carbonyl appearance and that the availability of Lys residues accounts for the
reduction of carbonylcontent at later time (presumably reflecting cross-linking chemistry) that occurs for acrolein and 4-oxo-2-nonenal. Overall, these results suggest that DNPH reactivity observed physiologically on nonmetalloproteins may arise more from the attachment of lipid-derived products of oxidative stress than fromadventitious MCO of side chains. Additional studies carried out to clarify the potential use of DNPHderivatization to tag peptide-based carbonyls for mass spectrometric analysis revealed that DNPHderivatization can reverse under the conditions used for proteolysis.