Oxidation Products Of Methionine Research Articles

386 Association between myeloperoxidase activity and methionine oxidation products in bronchoalveolar lavage and risk of bronchiectasis in infants and toddlers with cystic fibrosis

386 Association between myeloperoxidase activity and methionine oxidation products in bronchoalveolar lavage and risk of bronchiectasis in infants and toddlers with cystic fibrosis

Dissecting the Adaptive Response to Arginine Deprivation in Hodgkin Lymphoma

Background In Hodgkin Lymphoma (HL), neoplastic cells orchestrate an inflammatory microenvironment leading to sterile inflammation, T-cell anergy, and immune deficiency. Our group showed that in HL patients the aminoacid degrading enzyme Arginase-1 is increased, associated with poor outcome, and leads to arginine (Arg) deprivation. However, how the reduction of Arg in the extracellular milieu of the tumor microenvironment can contribute to neoplastic cell fitness is largely unknown.Aims To detect the adaptive response (via evaluation of global transcriptome and metabolome changes) in human HL cell lines exposed to Arg deprivation.Methods To better understand the impact of extra-cellular Arg1 deprivation on the metabolome of human cHL cells, four human cHL cell lines (L428, L540, HDMYZ and KM-H2) were individually cultured with customized complete media or lacking or Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments. After 48 hours of culture, the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by Illumina platform. Following normalization to DNA concentration, log transformation, and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups.Results While Arg deprivation did not affect cell viability but delayed cell cycle due to arrest in G2 phase in all tested cell lines, the effect of Arg deficiency on the cellular metabolome depended largely on the cell type examined with L428 and KMH2 cells having significantly changed metabolomes. Pyruvate was significantly higher in the KMH2 cells deprived of Arg compared to controls. Conversely, lactate was significantly lower, with increased levels of long-chain saturated fatty acids and long-chain polyunsaturated fatty acids (PUFAs). Taken together the metabolomics changes suggested that specific-amino acid deficiency can lead to an increase in free fatty acids synthases to preserve cytoplasmatic and mitochondrial membrane dynamics. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione in KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine).Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response (UPR, including XBP1, EIF2S1, EIF4A2, EIF4A3, ATF3, ATF4, DDIT4, EDEM1, GADD45B, SQSTM1, HMOX), NF-kB response to TNF (including RAF1, TNF, LIF, NKBIA, SGK1, BIRC3, ICAM1, BCL6, IL6, RELA, CDKN1A), p53 pathway and networks (including CDKN2B, STOM, TRAF4, RRAD, SESN1, FOXO3, SERPINB5, JAG2) and proteosome degradation (HSPA4, PSMD11, PSMD13, PSMD2, PSMA5, PSMA7, PSMC4), with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation.All lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, suggesting that early response to arginine deprivation includes modulation of UFMylation pathway, the most recently discovered post-translational protein modification system, whose biological function is largely unknown.Conclusions The removal of Arg from L428 and KMH2 resulted in changes in the specific-amino acid-related metabolites. The adaptive response to Arg-depleted environment increases oxidative stress and promotes a shift in glucose use in the attempt to preserve mitochondrial function. The cell-cycle arrest in G2, the increase of pyruvate availability and the upregulation of proteasome function via upregulation of the UFMylation pathway suggest the dependency of HL cell lines on mitochondrial function integrity. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. DisclosuresMartinelli: Stemline Therapeutics: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Abbvie: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy. Di Raimondo: Pfizer: Honoraria; Jazz Pharmaceutical: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

An Integrative Unbiased Global Analysis of the Metabolic Adaptive Response to Tryptophan Deprivation in Classical Hodgkin Lymphoma

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

Voltammetric and mass spectrometry investigation of methionine oxidation

The electro-oxidation mechanism of free methionine and bound within different peptide sequences was investigated by voltammetry, at glassy carbon electrode, and mass spectrometry. It is proposed that the electro-oxidation of free methionine occurs in two steps, each involving the transfer of one electron and turns pH-independent from mild acid to mild alkaline electrolytes. The first oxidation reaction leads to the formation of a cation radical stabilized either through the amino group resulting in the dehydromethionine intermediate, or by interaction with a neutral methionine molecule leading to production of a dimer cationic radical. The dehydromethionine hydrolysis gave methionine sulfoxide as final oxidation product, whereas a future oxidation of methionine dimer cation radical, i.e. the second electro-oxidation step, results in a methionine dimer dication. Moreover, at high acid media, the protonated amino group influence the electro-oxidation process to take place via proton transfer mechanism. The presence of methionine sulfoxide and of the dimer cationic radical as oxidation products of methionine was confirmed by mass spectroscopy.

The Oxidized Protein Repair Enzymes Methionine Sulfoxide Reductases and Their Roles in Protecting against Oxidative Stress, in Ageing and in Regulating Protein Function

Cysteine and methionine residues are the amino acids most sensitive to oxidation by reactive oxygen species. However, in contrast to other amino acids, certain cysteine and methionine oxidation products can be reduced within proteins by dedicated enzymatic repair systems. Oxidation of cysteine first results in either the formation of a disulfide bridge or a sulfenic acid. Sulfenic acid can be converted to disulfide or sulfenamide or further oxidized to sulfinic acid. Disulfide can be easily reversed by different enzymatic systems such as the thioredoxin/thioredoxin reductase and the glutaredoxin/glutathione/glutathione reductase systems. Methionine side chains can also be oxidized by reactive oxygen species. Methionine oxidation, by the addition of an extra oxygen atom, leads to the generation of methionine sulfoxide. Enzymatically catalyzed reduction of methionine sulfoxide is achieved by either methionine sulfoxide reductase A or methionine sulfoxide reductase B, also referred as to the methionine sulfoxide reductases system. This oxidized protein repair system is further described in this review article in terms of its discovery and biologically relevant characteristics, and its important physiological roles in protecting against oxidative stress, in ageing and in regulating protein function.

Evidence for the Formation of Ozone (or Ozone-Like Oxidants) by the Reaction of Singlet Oxygen with Amino Acids

Antibodies or some amino acids, namely, cysteine, methionine, histidine, and tryptophan, were previously reported to catalyse the conversion of singlet oxygen (1O2) to ozone (O3). The originally proposed mechanism for such biological ozone formation was that antibodies or amino acids catalyse the oxidation of water molecules by singlet oxygen to yield dihydrogen trioxide (HOOOH) as a precursor of ozone and hydrogen peroxide (H2O2). However, because HOOOH readily decomposes to form water and singlet oxygen rather than ozone and hydrogen peroxide, an alternative hypothesis has been proposed; ozone is formed due to the reaction of singlet oxygen with amino acids to form polyoxidic amino acid derivatives as ozone precursors. Evidence in support of the latter hypothesis is presented in this article, in that in the presence of singlet oxygen, methionine sulfoxide (RS(O)CH3), an oxidation product of methionine (RSCH3), was found to promote reactions that can best be attributed to the trioxidic anionic derivative RS+(OOO−)CH3or ozone.

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.

A kinetic and mechanistic study on the oxidation of l-methionine and N-acetyl l-methionine by cerium(IV) in sulfuric acid medium

The kinetics of oxidation of l-methionine and N-acetyl l-methionine by Ce(IV) in sulfuric acid–sulfate media in the range of 288.1–298.1K has been investigated. The major oxidation products of methionine and N-acetyl l-methionine have been identified as methionine sulfoxide and N-acetyl methionine sulfoxide. The major oxidation products have been confirmed by qualitative analysis and boiling point. The reaction was first order with respect to l-methionine, N-acetyl l-methionine and Ce(IV). Increase in [H+], ionic strength and HSO4- did not affect the reaction rate. Under the experimental conditions, Ce4+ was the effective oxidizing species of cerium. Increase in dielectric constant of the medium decreased the reaction rate. Under nitrogen atmosphere, the reaction system can initiate polymerization of acrylonitrile, indicating the generation of free radicals. Activation parameters associated with the overall reaction have been calculated.

Conversion of methionine into homocysteic acid in heavily oxidized proteomics samples

Analysis of protein oxidation is necessary in numerous areas of biochemistry, including hydroxyl radical surface mapping, oxidative stress assays, and pharmaceutical stability testing. Mass spectrometry is one of the tools most often used to identify protein oxidation products, and previous studies have attempted to identify and characterize all of the major oxidation products detected by mass spectrometry for each amino acid residue. In this note, we present evidence that in heavily oxidized protein samples, such as those produced by hydroxyl radical surface mapping, a major oxidation product of methionine is homocysteic acid. The formation of homocysteic acid from methionine was previously unrecognized in other mass spectrometric analyses, and has important implications for the analysis of oxidized samples, as well as potential implications as to the functional consequences of methionine oxidation.

Alterations in mitochondrial and cytosolic methionine sulfoxide reductase activity during cardiac ischemia and reperfusion

During cardiac ischemia/reperfusion, proteins are targets of reactive oxygen species produced by the mitochondrial respiratory chain resulting in the accumulation of oxidatively modified protein. Sulfur-containing amino acids are among the most sensitive to oxidation. Certain cysteine and methionine oxidation products can be reversed back to their reduced form within proteins by specific repair enzymes. Oxidation of methionine in protein produces methionine- S-sulfoxide and methionine- R-sulfoxide that can be catalytically reduced by two stereospecific enzymes, methionine sulfoxide reductases A and B, respectively. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during conditions of oxidative stress, the fate of this system during ischemia/reperfusion was investigated. Mitochondrial and cytosolic methionine sulfoxide reductase activities are decreased during ischemia and at early times of reperfusion, respectively. Partial recovery of enzyme activity was observed upon extended periods of reperfusion. Evidence indicates that loss in activity is not due to a decrease in the level of MsrA but may involve structural modification of the enzyme.

Effects of sucrose on rFVIIa aggregation and methionine oxidation.

The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from the protein's surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon incubation. Unlike aggregation, methionine oxidation was found to increase in the presence of sucrose. The two methionine residues susceptible to oxidation are presumably located at the protein surface, and the chemical potential increase in the presence of sucrose may account for the increase in oxidation rate. While FTIR spectroscopy suggested that sucrose induces small conformational changes in the rFVIIa structure, CD spectroscopy did not support this finding. The secondary structure of precipitated rFVIIa was changed when compared to the native solution secondary structure. Appearance of bands characteristic of intermolecular beta-sheet structure were found coincident with a decrease in alpha-helix and intramolecular beta-sheet structure.

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [ Me- 13C, Me- 2H 3]methionine sulfoxide and [ Me- 13C, Me- 2H 3]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-( tert-butyldimethylsilyl)- N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0±1.0 μM (means ± SD, n=8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.

Identification of tryptophan oxidation products in bovine alpha-crystallin.

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.

Activation of the fifth component of human complement, C5, without cleavage, by methionine oxidizing agents

Purified human C5 was incubated with chloramine T (Cl-T) or N-chloro-succinimide (N-Cl-S) in barbital buffer, pH 7.2. The treatment led to C5 activation: Cl-T- and N-Cl-S-treated C5 acquired a binding site for C6; upon incubation with C6 and subsequent addition of C7, C8 and C9 a membrane attack complex formed which lysed non-sensitized guinea pig red cells (reactive lysis). While the physiological activation of C5 follows its specific cleavage, the resulting fragment C5b representing the activated C5 and expressing the C6 binding site, the treatment with the mentioned chemicals does not lead to fragmentation of the C5 protein. So, functionally, the product of the chemical treatment is C5b-like, but chemically, it comprises the whole protein; no C5a is released. Cl-T and N-Cl-S are known to more or less selectively oxidize methionine residues in proteins, dependent on the conditions. Other sensitive amino acid residues are tryptophan and cysteine. Conditions were chosen for treatment of C5 with Cl-T which exclude attack on tryptophan, and we have ensured that human C5 does not contain free cysteine residues. Further, oxidation of about 60% of the methionine residues of C5 by Cl-T was demonstrated by amino acid analysis. So, all evidence points to methionine residue(s) as the site of attack of Cl-T and probably also of N-Cl-S. The oxidation product of methionine, its sulphoxide, may cause a change in structural conformation of C5 which involves expression of the C6 binding site. Earlier it was found that oxidation of C5 by hydroxyl radicals leads to its activation without cleavage. Since the properties of this C5b-like product resemble those of the product of treatment with Cl-T and N-Cl-S, it is suggested that the formerly found activation of human C5 by hydroxyl radicals is also mediated by oxidation of methionine residue(s) in the C5 protein.

Isolation of Oxidation Products of Methionine Formed under the Condition of Active Oxygen Method

Lard with methionine was oxidized under the active oxygen method (AGM). The oxidation products of methionine were investigated. Two oxidation products of methionine were isolated and determined by IR, NMR and synthetic identification as methionine sulfoxide and methionine sulfone, respectively.Antioxidative effect of methionine was attributed to the decomposition of the peroxide of lard to give the sulfoxide and sulfone.

Peptide characterization with a sulfoethyl aspartamide column

A strong cation-exchange (SCX) high-performance liquid chromatography column (sulfoethyl aspartamide, 200 × 4.6 mm) was used to analyze more than 50 peptides, ranging in length from 5 to 20 residues. These data show that the elution positions of the peptides increase monotonically with the number of positively charged residues. [A 60-min linear gradient of 0 to 100% eluent B at 1 ml/min was used, where eluent A is 5 m M phosphate (pH 3.0)-acetonitrile (75:25) and eluent B is eluent A + 0.5 M sodium chloride.] A comparison of SCX with a standard C 18 reversed-phase (RP) column [60-min linear gradient of 0 to 60% B at 1 ml/min, where eluent A is 0.1% trifluoroacetic acid (TFA), and eluent B is 0.095% TFA-acetonitrile (10:90)] further demonstrates the utility of SCX in peptide characterization. SCX separated an (Arg) 3-containing peptide from the Arg-deleted peptide while RP could not. In addition, SCX and RP resolved the methionine oxidation products of ACTH (4–10) (RP: Met [O]<Met [O 2]<Met; SCX: Met [O]<Met<Met [O 2]), suggesting a mixed-mode mechanism for the ion-exchange system. Finally, SCX separated the sulfated and non-sulfated forms of cholecystokinin (26–33) and Leu-enkephalin as well as the N-terminal acetylated forms of neurotensin (8–13) and angiotensinogen (1–14) from the respective unmodified peptides.

Utilization of L-Methionine Sulfoxide by the Rat

Methionine sulfoxide is an expected oxidation product of methionine in foods which undergo lipid oxidation or are treated with strong oxidizing agents like hydrogen peroxide. The extent to which methionine sulfoxide could replace methionine in the diet has therefore been tested using diets which are based upon mixtures of synthetic amino acids. This technique allows the exclusive and controlled testing of methionine sulfoxide, which previous studies using proteins could not achieve. The ability of the young rat to utilize the sulfoxide improves with its age, or alternatively there is a process of adaptation to permit more efficient utilization of the sulfoxide. An unexplained increase in the “methionine-like” content of blood plasma accompanies the ingestion of the sulfoxide.