Evaluation of different methods for assaying protein carbonylation

Most current methods for determining protein carbonyl content are based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenylhydrazone derivatives, which can then be detected and quantitated spectrophotometrically or immunochemically. In this article, we evaluate different quantitative and semi-quantitative techniques for measuring protein carbonylation. We assessed the sensitivity and accuracy of these techniques using both test samples and tissue extracts. We found that absorbance based quantitation of protein phenylhydrazone content is prone to experimental variation, often generating unreliable results. A previously described microplate ELISA method enabled very sensitive and accurate measurement of the carbonyl content of mixtures of reduced and oxidized BSA but proved to be quite insensitive when complex biological samples were assayed. Immunochemical detection and quantitation of DNPH derivatized samples spotted onto nitrocellulose membrane or processed using unifilter devices often produced inconsistent results. The difficulties observed with each of these methods appear to be associated with the presence of unreacted DNPI-I and nonprotein carbonyl. We found that short SDS-PAGE of derivatized sample efficiently removes these confounding substances and enables sensitive and reliable immunochemical detection of protein carbonyl after electrophoretic transfer of the proteins onto nitrocellulose membrane.