Publisher Summary This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.

Determination of carbonyl content in oxidatively modified proteins.

Beckman, Kenneth B., and Bruce N. Ames. The Free Radical Theory of Aging Matures. Physiol. Rev. 78: 547–581, 1998. — The free radical theory of aging, conceived in 1956, has turned 40 and is rapidl...

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The Free Radical Theory of Aging Matures

A number of systems that generate oxygen free radicals catalyze the oxidative modification of proteins. Such modifications mark enzymes for degradation by cytosolic neutral alkaline proteases. Protein oxidation contributes to the pool of damaged enzymes, which increases in size during aging and in various pathological states. The age-related increase in amounts of oxidized protein may reflect the age-dependent accumulation of unrepaired DNA damage that, in a random manner, affects the concentrations or activities of numerous factors that govern the rates of protein oxidation and the degradation of oxidized protein.

https://science.sciencemag.org/content/sci/257/5074/1220.full.pdf

Protein oxidation and aging

Protein carbonyl groups as biomarkers of oxidative stress.

We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins.

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Publisher Summary Specific oxidation of one or more methionine residues in a number of methionine-containing peptides, proteins, and enzymes has been recognized as an important regulatory principle, including control of chemotaxis, metabolic pathways, and sequence-independent recognition of nonpolar protein surfaces. Analysis of protein-bound methionine sulfoxide in tissue samples or body fluids from inflammatory sites and the correlation of this parameter with functional and structural changes appear to be a promising way to assess oxidative tissue injury. To quantify the relative methionine sulfoxide content in proteins, methionine and methionine sulfoxide must be determined simultaneously by amino acid analysis. However, a direct analysis of methionine sulfoxide in acid hydrolysates is subject to considerable error. Methionine is oxidized to methionine sulfoxide when the methionine sulfoxide content is low in comparison to the total methionine in proteins. The indirect method, using alkylation followed by performic acid oxidation, sometimes results in an underestimation of methionine sulfoxide, especially for proteins containing small amounts of the oxidized amino acid. The analysis of protein-bound methionine sulfoxide according to the cyanogens bromide procedure presented in the chapter includes (1) appropriate sample preparation, (2) quantitative conversion of nonoxidized methionine to homoserine and homoserine lactone, (3) acid hydrolysis under reducing conditions, (4) a quantitative conversion of homoserine lactone to homoserine, and (5) a simultaneous analysis of homoserine and residual methionine by reversed-phase high-performance liquid chromatography (HPLC) after precolumn derivatization with o-phthaldialdehyde.

Anke-G. Lenz

Papers

Determination of carbonyl content in oxidatively modified proteins.

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Analysis of methionine sulfoxide in proteins.