Because proteins are major components of biological systems and because proteins play an important role in a variety of cellular functions such as signal transduction, mitosis, cellular transport systems, chaperone activity, etc., an age-related increase in oxidative damage to proteins could be important physiologically to an organism. Stadtman’s laboratory was the first group to show that proteins were oxidatively damaged in vivo (Stadtman, 1990), and the physiological importance of oxidative damage to protein was recognized demonstrated over 30 years ago when Levine (1983) showed the oxidative modification of one of the histidine residues of glutamine synthetase to a carbonyl group inactivated the enzyme. Currently, protein oxidation is considered to be a potentially important marker for oxidative stress and is considered an important factor in various diseases as well as in aging.
Almost all amino acids in proteins are potential targets for oxidation by ROS (Huggins et al., 1993). For example, phenylalanine and tryptophan are converted to hydroxyl derivatives, tyrosine is converted to nitrotyrosine, and histidine is converted to 2-oxohistidine by oxidation. Methionine is unique in that its oxidation to methionine sulfoxide is reversible and can be converted back to methionine by methionine sulfoxide reductase. Cysteine can be oxidized to a series of products, which also include reversibly oxidized forms (disulfide, S-thiolated, S-nitrosylated, and sulfenic acid) and irreversibly oxidized forms (sulfinic and sulfonic acids). However, the most widely studied product of protein oxidation is the formation of carbonyl derivatives to side chains of certain amino acids, e.g., lysine, arginine, praline, and cysteine (Amici et al., 1989). Lipid peroxidation products and advanced glycation end products also can react with proteins forming carbonyl derivatives to the side chains of lysine and cysteine by the Michael reaction (Doorn and Petersen, 2002).
Stadtman’s laboratory developed a spectrophotometric based assay to detect protein carbonyls in proteins in biological samples (Levine et al., 1990). The assay uses 2,4,-dinitrophenylhydrazine (DNPH), which reacts with carbonyl groups and has an absorbance maxima at 370nm and a molar extinction coefficient of 22000 M-1 cm-1. The level of carbonyl groups in proteins is determined by the absorbance at 370nm associated with the acid precipitable material after reacting with DNPH. Using this assay, Stadtman’s group were the first to show that protein oxidation increased with age (Oliver et al., 1987). Over the past 30 years, the effects of aging and various pathological processes on the levels of carbonyl groups in protein extracts from tissues of laboratory rodents and human subjects have been studied using this assay (see Bokov et al., 2004 for a review). However, the major obstacle in the spectroscopic measurement of protein carbonyl content by the DNPH assay is the interference from the unreacted, free DNPH associated with the acid precipitates (Goto et al., 1999). Subsequently, assays using Western blots have been developed to identify proteins showing increased levels of carbonyl groups. Carbonyl groups are derivatized with DNPH or other groups and the derivatives are identified using antibodies directed against DNPH (Nakamura and Goto, 1996). However, the sensitivity of this assay is limited by the limited specificity of the available antibodies to DNPH-proteins.
To address this issue,our laboratory developed an assay to quantify protein carbonyl groups in proteins by derivatization of carbonyl groups with a fluorescent probe, fluorescein-5-thiosemicarbazide (FTC) (Chaudhuri et al., 2006). The hydrazine group on FTC reacts with the carbonyl to form a Schiff base covalently linking the fluorescein moiety to the protein. The fluorescence associated with the acid-precipitable protein pellets is used to measure the level of protein carbonyls fluorometrically with an excitation wavelength of 492 nm and emission wavelength of 516 nm. We showed the reaction of FTC with protein carbonyls was specific for carbonyl groups. However, the inability to remove the free FTC from acid-insoluble material by washing resulted in a great deal of variation, and we found that it was virtually impossible to detect with any consistency an increase in protein carbonyl groups formed in vitro or in vivo after an oxidative stress. Therefore, we resolved the proteins on SDS-polyacrylamide gel electrophoresis, which separated the free FTC from FTC covalently bound to the oxidized proteins. Using gel electrophoresis, we are able to determine the carbonyl content (nmoles/mg of protein) of the proteins by measuring the fluorescence associated with the proteins on the gels. As shown in the Figure below, the protein carbonyl levels increased 2-fold in the liver of old mice compared to young mice. The major advantage of the FTC labeling assay is that it allows one to directly measure changes in the levels of carbonyl groups in specific proteins using 2-D gel electrophoresis as shown in the Figure. The current methodology used to measure changes in carbonyl groups in specific proteins employs antibodies to the DNP-protein adducts (Nakamura and Goto, 1996). However, an accurate quantification of protein levels by western blots is limited by several factors including the specificity of the antibodies for the DNP-protein adducts and non-quantitative transfer of proteins from SDS-polyacrylamide gel into the PVDF membrane. In contrast, the FTC assay allows one to directly determine the levels of carbonyl groups in specific proteins by measuring the fluorescence associated with each spot. We have shown that FTC-bound proteins are neither lost during 2D gel electrophoresis nor show a differential migration on 2-D gels compared to the proteins free of FTC. An additional advantage of the FTC labeling assay is that one can measure on the same gel both the incorporation of FTC into each protein and the level of protein in the spot using Sypro Ruby. Thus, by measuring the ratio of the fluorescence intensities of FTC and Sypro Ruby, one can calculate the pmoles of carbonyl/µg of protein. Measuring this ratio is important for several reasons. First, it allows one to minimize gel to gel variation because of unequal loading of proteins on the gel. Second, it allows one to more directly compare changes in the carbonyl content of abundant to lesser abundant proteins. Third, the ratio allows one to eliminate the possibility that the changes in carbonyl content are secondary to changes in the expression of protein, i.e., either decreased or increased levels of the specific protein in the extract. Finally, one can calculate the moles of carbonyl groups/mole of protein from this ratio once the protein is identified. In other words, it is possible to determine the molar carbonyl content in specific proteins.
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