Use of modified CUPRAC and dinitrophenylhydrazine colorimetric methods for simultaneous measurement of oxidative protein damage and antioxidant defense against oxidation

Abstract

A modified CUPRAC (cupric reducing antioxidant capacity) method was developed for the simultaneous estimation of protein oxidation and counteracting antioxidant defense, and the results were compared with those of a modified 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay. The alkaline carbonyl method was cleared off interferences by solvent extraction using a cationic surfactant. Both solution and Nafion membrane sensor CUPRAC methods were used to measure the oxidative hazard in protein solutions. Bovine serum albumin, fetal bovine serum and egg white were used as protein probes, exposed to oxidation by Fe(II)-induced Fenton reaction in the absence and presence of selected antioxidants (ascorbic acid, cysteine, gallic acid, glutathione, and N-acetyl cysteine). Protein probes were initially unreactive toward the CUPRAC and DNPH reagents, but produced colored products upon Fenton oxidation which were bleached by antioxidants, enabling an indirect measurement of antioxidant activity (AOA) by difference. Spearman's rank test for antioxidants demonstrated that there was a strong correlation (+0.7 to +0.9) between the modified CUPRAC and carbonyl assays. There was also a strong correlation between the results of the solution phase and optical sensing CUPRAC methods (R2 > 0.95). As opposed to conventional antioxidant assays not using biologically relevant probes, this work utilizes protein probes for AOA assessment.