Spectrophotometric assay of protein carbonyls in human plasma

Abstract

Introduction: The oxidative stress represents the aggression produced at the molecular level by the imbalance between pro-oxidant and antioxidant agents, with severe functional consequences in all organs and tissues. An overproduction of reactive oxygen species (ROS) results in oxidative damages especially in proteins (the main target of ROS), as well as in lipids, or DNA. A great effort has been undertaken to assess the biomarkers of protein oxidative injury, most cited being the quantitative assay of protein carbonyls, nitrotyrosine levels, GSH level and/or GSH/GSSH ratio. The present study aims at finding a robust spectrophotometric method to quantify the protein carbonyls in human plasma. The study model was represented by bovine serum albumin (BSA), which was subjected to different oxidative damage conditions, in order to be carbonylated. Materials and methods: Three hydroxyl radical generating systems were investigated: potassium ascorbate / ferric chloride, ascorbic acid / ferrous sulfate and hydrogen peroxide / copper sulfate. All systems were applied to BSA solutions for 10 to 24 h at 37 Celsius degrees. After degradation, the protein carbonyl levels were evaluated by means of a modified Levine’s method, using the derivatisation with 2,4-dinitrophenylhydrazine. The carbonyl content was expressed in mg carbonyls/mg proteins. An ABL&E-JASCO model V 530 spectrophotometer was used throughout the experiments. Results: The best yield for carbonyl generation was induced by potassium ascorbate / ferric chloride system, after 24 h degradation time. The carbonyl proteins pellets are separated only at centrifuge speeds higher than 5000 rpm, with an optimum at 7000 rpm, at 4 Celsius degrees. The protein carbonyls are stable in the BSA solution only for a short period (hours). Levine's method was modified by replacing the solubilisation in guanidine hydrochloride with 1 M NaOH solution. The total protein content was assessed using Lowry method, in order to avoid nucleic acids interferences. The method was validated according to the EM EA/CHM P/EW P/192217/2009 “Guideline for bioanalytical method validation”. Conclusion: A controlled method for the generation and a validated method for the quantification of protein carbonyls using BSA as a model, with spectrophotometric assay were developed. The methods can be further used for human plasma protein assay. Also, it could be useful for the development of a new capillary electrophoresis method for protein carbonyls assay.