A novel fluorimetric assay for visualization and quantification of protein carbonyls in muscle foods

Abstract

Muscle foods, particularly fish products are highly exposed to oxidative stress during processing and storage, resulting in oxidative modification of proteins. Protein carbonyls content has been used as one of the measures of oxidative stress. Generally, the resulting carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and detected with anti-DNP antibody. However, the applicability of this method to food samples is limited by its high price, time-consuming procedure and possibility to perform the measurements just on soluble protein fractions. We developed a simpler, faster and cheaper method to assess CP level in muscle foods, including both soluble and insoluble protein fractions, which is based on a direct reaction of protein carbonyls with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The paper describes a novel technique to label both soluble and insoluble carbonylated proteins with CHH and determine carbonyl content by fluorescence microscopy assay which correlates (R = 0.911) with conventional ELISA method.

A novel fluorimetric assay for visualization and quantification of protein carbonyls in muscle foods