Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.     

Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix-assisted laser desorption/ionization mass spectrometry

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

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.     

Modulating protein activity and cellular function by methionine residue oxidation

The sulfur-containing amino acid residue methionine (Met) in a peptide/protein is readily oxidized to methionine sulfoxide [Met(O)] by reactive oxygen species both in vitro and in vivo. Methionine residue oxidation by oxidants is found in an accumulating number of important proteins. Met sulfoxidation activates calcium/calmodulin-dependent protein kinase II and the large conductance calcium-activated potassium channels, delays inactivation of the Shaker potassium channel ShC/B and L-type voltage-dependent calcium channels. Sulfoxidation at critical Met residues inhibits fibrillation of atherosclerosis-related apolipoproteins and multiple neurodegenerative disease-related proteins, such as amyloid beta, α-synuclein, prion, and others. Methionine residue oxidation is also correlated with marked changes in cellular activities. Controlled key methionine residue oxidation may be used as an oxi-genetics tool to dissect specific protein function in situ.     

Methionine oxidation and aging

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.     

Characterization of structural unit of phospholamban by amino acid sequencing and electrophoretic analysis

The partial amino acid sequence of phospholamban from canine cardiac sarcoplasmic reticulum was determined by sequence analysis of the peptides obtained from the protein cleaved by cyanogen bromide and with TPCK-trypsin. The sequence determined initiated with N alpha-acetylated methionine followed by 44 amino acid residues intervening two unidentified residues. This polypeptide would represent a structural unit (protomer) of phospholamban. Analysis of temperature-dependent conversion of phospholamban from 26 kDa to lower molecular weight form (6 kDa) suggested that phospholamban holoprotein is composed of five identical protomers.     

Protection of GroEL by its methionine residues against oxidation by hydrogen peroxide

GroEL undergoes an important functional and structural transition when oxidized with hydrogen peroxide (H2O2) concentrations between 15 and 20mM. When GroEL was incubated for 3h with 15 mM H2O2, it retained its quaternary structure, chaperone and ATPase activities. Under these conditions, GroEL's cysteine and tyrosine residues remained intact. However, all the methionine residues of the molecular chaperone were oxidized to the corresponding methionine-sulfoxides under these conditions. The oxidation of the methionine residues was verified by the inability of cyanogen bromide to cleave at the carboxyl side of the modified methionine residues. The role for the proportionately large number (23) of methionine residues in GroEL has not been identified. Methionine residues have been reported to have an antioxidant activity in proteins against a variety of oxidants produced in biological systems including H2O2. The carboxyl-terminal domain of GroEL is rich in methionine residues and we hypothesized that these residues are involved in the protection of GroEL's functional structure by scavenging H2O2. When GroEL was further incubated for the same time, but with increasing concentrations of H2O2 (>15 mM), the oxidation of GroEL's cysteine residues and a significant decrease of the tyrosine fluorescence due to the formation of dityrosines were observed. Also, at these higher concentrations of H2O2, the inability of GroEL to hydrolyze ATP and to assist the refolding of urea-unfolded rhodanese was observed.     

Determination of methionine sulfoxide in protein and food by hydrolysis with p-toluenesulfonic acid

Methionine sulfoxide in peptides and proteins was determined by use of 3 N p-toluenesulfonic acid as a hydrolyzing agent. Samples were hydrolyzed at 110 degrees C for 22 h in an evacuated sealed tube and analyzed for amino acid content. Amino acid analysis showed that the recovery of methionine sulfoxide from a synthetic peptide and its mixture with proteins was consistently better than 90%. The recovery of all other amino acids except tryptophan was complete, and was similar to that observed after hydrolysis with 6 N HCl. The presence of carbohydrates had no effect on the yield. Thus, the present procedure can be used for general and simultaneous determination of methionine sulfoxide as well as other amino acids in proteins.     

The amino acid sequence of thiogalactoside transacetylase of Escherichia coli

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.     

The complete amino acid sequence of the beta-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

The complete amono aicd sequence of the beta-subunit of protocatechuate 3,4-dioxygenase is presented. The beta-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated beta-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphyloccal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated beta-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Amino acid sequence of Streptomyces metallo-proteinase inhibitor from Streptomyces nigrescens TK-23

Streptomyces Metallo-Proteinase Inhibitor (S-MPI) consists of 102 amino acid residues, including one methionine and two disulfide bridges. The complete amino acid sequence of S-MPI, including two disulfide bridges, was determined by sequencing of tryptic and chymotryptic peptides of two fragments obtained by cyanogen bromide cleavage followed by reduction and S-pyridylethylation of the protein. Incubation of the inhibitor with thermolysin slowly cleaved one peptide bond, Cys(64)-Val(65), which might be a reactive site of S-MPI.     

Determination of methionine sulfoxide in proteins: comparison of a gas-chromatographic and electrophoretic method

Two methods for the determination of methionine in proteins have been used to estimate the extent of methionine sulfoxide obtained upon exposure of proteins to oxidizing agents. Both methods are based on prior treatment with cyanogen bromide, which attacks methionines (but not the sulfoxide derivative) with the resultant formation of methyl thiocyanate and peptides. The amount of methyl thiocyanate is determined quantitatively by gas chromatography, while the number of peptides is ascertained by SDS-polyacrylamide gel electrophoresis. The gas chromatographic estimate of CH3SCN offers an accurate and precise method (down to nanogram values) for the quantitative determination of methionine sulfoxide in proteins. Due to its simplicity and the use of low-cost equipment, the electrophoretic method appears to be a valuable complement to the gas chromatographic method, and the two methods in conjunction provide novel results.     

Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides

Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCl at room temperature. Other sulfoxides can be used in combination with HCl; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of methionine and oxindolylalanine. Methionine and cysteine are quantitatively oxidized by DMSO/HCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides LRF (luteinizing hormone-releasing factor), somatostatin, valine-gramicidin A and ACTH 1-24 were each treated with the DMSO/HCl reagent in acetic acid solution and the corresponding oxindolylalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis of ultraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content of acid hydrolyzates of the oxidized peptides with 3N-p-toluenesulfonic acid at 110 degrees for 24 h. The results indicate that modification of tryptophan peptides with DMSO/HCl provides a useful procedure, which seems superior to previously used reagents. In addition, the method could be well applied to other indoles of biological and pharmacological interest.     

Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.     

Use of N-chlorosuccinimide/urea for the selective cleavage of tryptophanyl peptide bonds in proteins. Cytochrome c.

The conditions and utility of the N-chlorosuccinimide/urea (NCS/urea) reagent for the selective cleavage of tryptophanyl peptide bonds in proteins is demonstrated with cytochrome c. At low concentrations of NCS/urea the oxidation of thioether side chains in cytochrome c is the predominant reaction. Methionyl residues are oxidized to sulfoxide and the heme-thioether bridge is partially cleaved. At 10-fold excess of NCS/urea reagent, cleavage of the tryptophanyl peptide bond is optimal at approximately 50% yield in several species of cytochrome c studied. Analytical data on isolated horse cytochrome c peptide fragments demonstrate lack of modification and cleavage at tyrosyl and histidyl residues. However, at high concentrations of NCS/urea reagent (30-fold) unexpected conversions of methionine to sulfone and cysteine to cysteic acid in intact proteins are observed. This is in contradistinction to the absence of sulfone in NCS/urea-reacted amino acid mixtures. The mechanisms of halogenation and cleavage by N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide are discussed. It is porposed that the selectivity with respect to halogenation by N-chlorosuccinimide is due to the insignificant participation of molecular chlorine in the NCS/urea reaction. A mechanism of halogenation and cleavage by NCS at tryptophan is also offered.     

Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.     

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.