Earl R. Stadtman

DETERMINATION OF CARBONYL CONTENT IN OXIDATIVELY MODIFIED PROTEINS

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.

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

Summary. 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.

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.

Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.

Protein Oxidation in Aging and Age‐Related Diseases

Abstract: Although different theories have been proposed to explain the aging process, it is generally agreed that there is a correlation between aging and the accumulation of oxidatively damaged proteins, lipids, and nucleic acids. Oxidatively modified proteins have been shown to increase as a function of age. Studies reveal an age‐related increase in the level of protein carbonyl content, oxidized methionine, protein hydrophobicity, and cross‐linked and glycated proteins as well as the accumulation of less active enzymes that are more susceptible to heat inactivation and proteolytic degredation. Factors that decelerate protein oxidation also increase the life span of animals and vice versa. Furthermore, a number of age‐related diseases have been shown to be associated with elevated levels of oxidatively modified proteins. The chemistry of reactive oxygen species‐mediated protein modification will be discussed. The accumulation of oxidatively modified proteins may reflect deficiencies in one or more parameters of a complex function that maintains a delicate balance between the presence of a multiplicity of prooxidants, antioxidants, and repair, replacement, or elimination of biologically damaged proteins.

Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli.

Abstract The glutamine synthetase of Escherichia coli, which is subject to cumulative feedback inhibition by eight different products of glutamine metabolism, is also regulated by repression. The enzyme is repressed by growth on media containing readily available nitrogen, such as yeast extract-peptone or high concentrations of ammonium salts. A twentyfold derepression occurs with growth-limiting concentrations of ammonia or by growth on glutamate as the sole nitrogen source. In contrast, the level of glutamate dehydrogenase is high after growth on elevated ammonium salt levels and is low with growth on glutamate. Glutamine synthetase from derepressed cells has been purified 180-fold, and was obtained as a crystalline protein which is homogeneous in the ultracentrifuge and by disc gel electrophoresis. Apparent Km values were determined for the various substrates and activators involved in the synthesis of glutamine from ATP, glutamate, and NH3 as well as for the enzyme-catalyzed transfer of the γ-glutamyl group from glutamine to hydroxylamine. Divalent cation activator effects were examined with respect to the pH optimum of the enzyme and utilization of substrate. Whereas Mn++ is specifically required in the transfer reaction when high levels of hydroxylamine are used, Mg++ will replace Mn++ as an activator in the overall reaction resulting in glutamine formation. The ability of analogues of glutamate, ATP, ADP, and ammonia to replace the normal substrates has been explored. In contrast to glutamine synthetases from other sources, the E. coli enzyme exhibits marked specificity for the l isomer of glutamate. Analytical ultracentrifugation, amino acid analyses, manganese determination, and light-scattering measurements have been performed, and indicate that the enzyme has a molecular weight of 680,000. Glutamine synthetase may be disaggregated into 12 or 14 subunits, this occurring in the presence of 5 m guanidine, or 1 m urea and 0.01 m EDTA. Fingerprint analyses of tryptic peptides of the enzyme suggest that all of the subunits are identical.

Oxidative modification of proteins during aging

Accumulating experimental evidence supports the proposal that many of the changes which occur during aging are a consequence of oxidative damage. Reactive oxygen species react with all three of the major cellular macromolecules, nucleic acids, lipids, and proteins. This minireview focuses on proteins as targets of oxidizing species during aging. Many of the reactions mediated by these oxidizing species result in the introduction of carbonyl groups into proteins. The steady-state level of carbonyl-bearing proteins increases exponentially during the last third of lifespan in animals ranging from C. elegans to man. Genetic and non-genetic manipulations which lengthen lifespan cause a decrease in the level of protein carbonyl while those which shorten lifespan increase the level. Oxidized proteins bearing carbonyl groups are generally dysfunctional, and in the last third of lifespan the content of these oxidized proteins rises to a level likely to cause substantial disruption of cellular function.

Methionine residues may protect proteins from critical oxidative damage.

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure. In contrast, the key functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the sixteen methionine residues could be oxidized with little effect on activity. The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein. Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site. Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue. Given the importance of oxidative stress during aging, the potential function of methionine residues as antioxidants during aging should be investigated experimentally.

Oxidation of Methionine in Proteins: Roles in Antioxidant Defense and Cellular Regulation

The roles of methionine residues in proteins have not been well defined, but a review of available studies leads to the conclusion that methionine, like cysteine, functions as an antioxidant and as a key component of a system for regulation of cellular metabolism. Methionine is readily oxidized to methionine sulfoxide by many reactive species. The oxidation of surface exposed methionines thus serves to protect other functionally essential residues from oxidative damage. Methionine sulfoxide reductases have the potential to reduce the residue back to methionine, increasing the scavenging efficiency of the system. Reversible covalent modification of amino acids in proteins provides the mechanistic basis for most systems of cellular regulation. Interconversion of methionine and methionine sulfoxide can function to regulate the biological activity of proteins, through alteration in catalytic efficiency and through modulation of the surface hydrophobicity of the protein.

Oxidation of Methionine Residues of Proteins: Biological Consequences

Most reactive oxygen species (ROS) can oxidize methionine (Met) residues of proteins to methionine sulfoxide (MetO). However, unlike the ROS-dependent oxidation of other amino acid residues of proteins (except cysteine residues), the oxidation of Met residues is readily reversed by the action of methionine sulfoxide reductase (Msr) that catalyzes the thioredoxin-dependent reduction of MetO residues of proteins back to Met. We summarize here results of studies showing that the cyclic interconversion of Met and MetO residues of proteins is involved in several different biological processes: (a) It is the basis of an important antioxidant mechanism for the scavenging of ROS. (b) It is likely involved in the regulation of enzyme activities. (c) It is involved in cell signaling. (d) It can target proteins for proteolytic degradation. Furthermore, a loss in the ability to catalyze the reduction of protein MetO to Met residues leads to a decrease in the maximum life span, whereas overexpression of this activity leads to an increase in the life span of animals. In addition, a decrease in Msr activities in brain tissues is associated with the development of Alzheimers disease.