Determination of sulfur amino acids in foods as related to bioavailability

During the processing of feedstuffs and foods, methionine can be oxidized to methionine sulfoxide and methionine sulfone, and cysteine can be oxidized to cysteic acid. Methionine sulfone and cysteic acid are nutritionally unavailable, but methionine sulfoxide can be utilized, at least to some degree. The degree of utilization depends on the levels of methionine, cysteine, and methionine sulfoxide in the diet, but there is no consensus in the literature on the quantitative impact of these dietary constituents on methionine sulfoxide utilization. Methionine and cysteine are most often determined after quantitative oxidation to methionine sulfone and cysteic acid, respectively, using performic acid oxidation prior to hydrolysis. However, this method may overestimate the methionine content of processed foods, as it will include any methionine sulfoxide and methionine sulfone present. A selection of analytical methods has been developed to allow the separate determination of the 3 oxidized forms of methionine, the merits of which are discussed in this review. An additional consideration for determining methionine and cysteine bioavailability is that not all dietary methionine and cysteine is digested and absorbed from the small intestine. Selected methods designed to determine the extent of digestion and absorption are discussed. Finally, a concept for a new assay for determining methionine bioavailability, which includes determining the digestibility of methionine and methionine sulfoxide as well as the utilization of methionine sulfoxide, is presented.     

A contribution to the voltammetric study of cystine and cysteine at Pt electrodes in 0.5 M H2SO4

Both cysteine and cystine adsorb at the Pt electrode according the Frumkin—Temkin isotherm with the heterogeneity factor f = 51 for cysteine and 21 for cystine. Both the adsorbed cysteine and cystine give in a solution without any dissolved cystine or cysteine almost identical first cyclic voltammetric curves. Each substance dissolved in the electrolyte gives two oxidation peaks which differ when the oxidation is carried out at a “reduced” or an “oxidized” Pt electrode. On the basis of the dependence of the height and potential of the peaks on polarization rate and concentration (in the case of oxidation of dissolved substances) and of coulometric measurements the following conclusions have been made concerning the kinetics and mechanism:(i) Neither cysteine nor cystine change their oxidation state on adsorption at the electrode.(ii) The final oxidation product of both adsorbed cysteine and cystine may be the cysteic acid.(iii) For cysteine there are two adsorbed species, one strongly adsorbed, the other one weakly adsorbed.(iv) The oxidation of dissolved cysteine takes place via the weakly adsorbed species, the surface concentration of which is influenced by the coverage of the strongly adsorbed species. This process is described by an electrode reaction rate equation.(v) In the overall oxidation of cysteine one electron is transferred while the detailed mechanism requires an oxidation by splitting-off two electrons with a subsequent ion—substrate dimerization reaction.     

氮氧自由基的研究(Ⅳ)——氮氧自由基对半胱氨酸的氧化反应

氮氧自由基是目前最广泛采用的自旋标记物^[1-2]。2,2,6,6-四甲基哌啶氮氧自由基虽然在许多情况下是很稳定的,但是仍然可以发生歧化、单电子氧化还原等反应^[3-4]。     

高效液相色谱-柱前衍生化法测定饲料中的含硫氨基酸

发展了一种饲料中含硫氨基酸的检测方法。样品经过甲酸氧化,将其中的胱氨酸和蛋氨酸分别氧化为磺基丙氨酸和蛋氨酸砜;再经酸水解后,采用2,4-二硝基氟苯进行柱前衍生化,衍生物经高效液相色谱分析。使用Elite AAK C18色谱柱(250 mm×4.6 mm, 5 μm)分离;以0.05 mol/L乙酸钠和乙腈-水(50:50, v/v)为流动相,梯度洗脱,流速为1.2 mL/min;检测波长为360 nm;柱温为31 ℃。结果表明,胱氨酸在0.4～16.0 mg/L、蛋氨酸在0.7～29.6 mg/L范围内的线性相关系数分别为0.9999、0.9998;胱氨酸和蛋氨酸的定量限(信噪比(S/N)=10)分别为2.6 μg/kg和3.1 μg/kg;3次样品平行测定的胱氨酸和蛋氨酸含量的相对标准偏差(RSD)分别为0.79%和2.56%,加标回收率分别为100.28%～102.00%和105.72%～107.89%。该方法分析成本低,测定结果准确,灵敏度高,符合饲料中含硫氨基酸的检测要求。     

Boron doped diamond and glassy carbon electrodes comparative study of the oxidation behaviour of cysteine and methionine

The electrochemical oxidation behaviour at boron doped diamond and glassy carbon electrodes of the sulphur-containing amino acids cysteine and methionine, using cyclic and differential pulse voltammetry over a wide pH range, was compared. The oxidation reactions of these amino acids are irreversible, diffusion-controlled pH dependent processes, and occur in a complex cascade mechanism. The amino acid cysteine undergoes similar three consecutive oxidation reactions at both electrodes. The first step involves the oxidation of the sulfhydryl group with radical formation, that undergoes nucleophilic attack by water to give an intermediate species that is oxidized in the second step to cysteic acid. The oxidation of the sulfhydryl group leads to a disulfide bridge between two similar cysteine moieties forming cysteine. The subsequent oxidation of cystine occurs at a higher potential, due to the strong disulfide bridge covalent bond. The electro-oxidation of methionine at a glassy carbon electrode occurs in two steps, corresponding to the formation of sulfoxide and sulfone, involving the adsorption and protonation/deprotonation of the thiol group, followed by electrochemical oxidation. Methionine undergoes a one-step oxidation reaction at boron doped diamond electrodes due to the negligible adsorption, and the oxidation also leads to the formation of methionine sulfone.     

High‐throughput method for on‐target performic acid oxidation of MALDI‐deposited samples

An information‐rich on‐target performic acid oxidation method, which is compatible with alkylation for differentiation of free cysteine versus disulfide‐containing peptides, is described. On‐target oxidation is achieved using performic acid vapor to oxidize disulfide‐containing peptides and/or small proteins on the matrix‐assisted laser desorption/ionization (MALDI) sample deposits. The on‐target oxidation method is preferred over solution‐phase oxidation methods because (1) less sample handing is required, (2) oxidation throughput is drastically increased and (3) ion suppression effects are reduced because performic acid is not added directly to the MALDI spot. The utility of this method is demonstrated by simultaneous oxidation of multiple MALDI sample deposits containing model disulfide‐linked peptides, intact bovine insulin and a bovine ribonuclease A proteolytic digest. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative high-performance liquid chromatographic determination of sulfur amino acids in protein hydrolysates

A rapid, quantitative high-performance liquid chromatographic procedure for the determination of methionine and cystine after oxidation to methionine sulfone and cysteic acid is described. The Dns derivatives of the amino acids are separated by reversed-phase chromatography with a phosphate buffer-acetonitrile gradient and detected by UV absorption at 254 nm. The procedure is validated by confirming the methionine and cystine content of ribonuclease A. The average yields of cysteic acid and methionine sulfone from triplicate analyses of ribonuclease A were 98.1% (±3.3) and 106.1% (±2.4) of the theoretical values, respectively.     

Application de la chromatographie sur échangeur d'ions a l'étude de la composition des aliments en acides amines

A detailed procedure is presented for the determination of the amino acid composition of foods by chromatography of the hydrolysates on columns of Dowex-50 by the method of Moore and Stein. Nearly all of the common amino acids can be determined. The measurement of methionine, however, is complicated by partial oxidation of the amino acid to the sulfoxide during acid hydrolysis. A satisfactory chromatographic method for tryptophan has not been obtained as yet. Cross-reference is given to the auxiliary development of a Chromatographic method for the determination of cystine (in the presence of carbohydrate) as cysteic acid and to the utilization of these combined techniques in the analysis of several foods of importance in human and animal nutrition.     

FURTHER STUDIES ON THE CRYSTAL-VIOLET-SENSITIZED PHOTOOXIDATION OF CYSTEINE TO CYSTEIC ACID

Abstract —Crystal violet sensitizes the selective photooxidation of cysteine to cysteic acid; hydrogen peroxide is also formed as an end product. The participation of singlet oxygen in the photoreaction has been ruled out, since exposure of cysteine to this reagent, generated by chemical or photochemical processes, gives only cystine as a product. The photoreaction is inhibited by radical scavengers such as hydroquinone and allylic alcohol. A mechanism is proposed involving hydrogen abstraction by the triplet dye from the thiol group of cysteine.     

Age estimation of old carpets based on cystine and cysteic acid content

A method for the evaluation of the age of wool carpets and textiles was developed based on the age dependent alteration of amino acid composition of proteins. Samples of 23 wool carpets and textiles of known age, obtained from the Hungarian Museum of Industrial Arts and the Hungarian National Museum were analysed for amino acid content. Results were compared with data obtained for contemporary, untreated wool and wool carpet. The cysteic acid content of wool increases with age. The contemporary wool carpet contained 0.31 g of cysteic acid in 100 g of protein. Comparable figures were 1.87 g for a 550-year old carpet and 4.01–4.39 g for the 1600–1750 year old wool carpets. The cystine content decreased with age, the corresponding figures being 7.88, 3.12 and 1.19-0.97 g/100 g, respectively. Corresponding contents of methionine were 0.43, 0.21 and 0.20-0.00 g/ 100 g and for tyrosine 3.07, 2.11 and 0.20-0.00 g/100 g. Prediction equations were developed as linear regressions of the age of wool on cysteic acid, cystine and tyrosine contents. The 95% confidence intervals of estimates for two samples of unknown age were estimates plus or minus 30 and 38 years.     

New chiral derivatization reagent for the resolution of amino acids as diastereomers by TLC and HPLC

Summary The applicability of a new chiral reagent to the resolution of amino acid enantiomers has been investigated. The new reagent, S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarbonyl chloride (NSP-C1), was synthesized by the chlorination of S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarboxylic acid which was prepared by the reaction of 2-naphthalene sulphonyl chloride with L-proline. Derivatization of the amino acids proceeds rapidly at ambient temperature and no racemization takes place during the reaction. The resolution of the diastereomeric amides was performed by TLC and normal phase HPLC. Complete resolutions were observed for the enantiomers of all amino acids examined except cysteine, cystine and histidine. The favourable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.1% level.     

THE PHOTOLYSIS OF CYSTINE IN ACIDIC AQUEOUS SOLUTION

Abstract— Quantum yields of cysteine, ammonia, 1-amino,1'-oxo,2,2'-dithiodipropionicacid (AODT–DPA), alanine, alanine 3-sulphinic acid, cysteic acid, and serine have been determined in aqueous oxygenated and deaerated cystine solutions irradiated with 254 nm radiation. From the effect of methanol, ethanol and propanol-2 on the quantum yields of cysteine, ammonia, AODT-DPA and alanine, it is concluded that (a) the S–S bond is broken with high quantum efficiency, (b) C–S and C–N bonds do not undergo primary photolytic fission, and (c) all the AODT–DPA, but only about 12 per cent of the ammonia, is free-radical in origin. The production of pyruvic acid at the expense of AODT–DPA in irradiated cystine solutions containing alanine provides further evidence that AODT–DPA has free-radical precursors. Reaction schemes are proposed for the radical-induced production of keto acid and ammonia in oxygenated and deaerated solutions.     

Titrimetric determination of some organic compounds with bromine chloride

Bromine chloride is used in hydrochloric acid medium as a standard reagent for the rapid and precise determination of organic compounds by direct or indirect titrimetric methods. Hydrazine and its aryl derivative undergo a 4-electron change. Carbonyl compounds are determined by reaction with excess of 2,4-dinitrophenylhydrazine and estimation of the surplus. Sulphanilamide undergoes a substitution reaction in 1:3 molar ratio to bromine chloride. Thiobarbituric acid and thiourea or its alkyl derivatives show an 8-electron change but aryl thioureas also undergo nuclear bromination. Thiosemicarbazide and semicarbazide give a 10- and a 2-electron change respectively. In the direct titration, methionine is oxidized to its sulphoxide whereas cystine and cysteine form cysteic acid. In presence of bromide, glutathione forms the sulphonic acid but in the presence of iodide the product is the disulphide. The analytical results obtained by bromine chloride method are compared favourably with those afforded by established procedures.     

The structure of melanins and melanogenesis—III The structure of sepiomelanin

New degradation products of sepiomelanin have been obtained. Alkali fusion yields, in addition to other compounds, 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, 4-methyl-cathechol and a compound, which is probably 5,6-dihydroxyindole-4,7-dicarboxylic acid. These products constitute the first proof of the indole structure of a natural melanin. The carboxyl group of 5,6-dihydroxyindole-2-carboxylic acid is not formed during alkali fusion, but pre-exists in the macromolecule. Cysteic acid, taurine, glycine and aspartic acid were obtained by oxidation of sepiomelanin with hydrogen peroxide in acetic acid. The formation of cysteic acid indicates that sepiomelanin is bound to the protein by means of cysteine. Taurine is clearly an artifact generated by decarboxylation of cysteine. Glycine and aspartic acid probably are derived from the pyrrole moiety of the indole units: they also result from the oxidation of 5,6-dihydroxyindole-2-carboxylic acid.

Oxidation of methylated sepiomelanin yields 3-carbomethoxypyrrole-2,5-dicarboxylic acid and 5-carbomethoxypyrrole-2,3-dicarboxylic acid; isolation of the former further proves the presence of pyrrole units in sepiomelanin, whereas formation of the latter is further evidence that some indole (probably dopachrome) units of the macromolecule have a carboxyl group in position 2.     

L-磺基丙氨酸的电化学合成及表征

Ｌ 磺基丙氨酸是一种重要的生化试剂 ,在医药、化妆品、洗涤剂等行业应用广泛 ;目前主要是采用过氧化物、二甲亚砜、溴等氧化Ｌ 胱氨酸制备它[1 ,2 ] ,我们尝试采用间接电氧化法合成 ,其特点是在电化学合成时 ,氧化媒质循环利用 ,避免了使用较贵的氧化剂 ,省去了还原产物的后处理 ,工艺简单 ,成本低 ,产物纯度高 ,而且无“三废”排放 ,与化学合成法相比有明显优势。1　实验部分1 1　主要实验仪器Ｍ 1 730红外光谱仪 ;ＦＸ90Ｑ核磁共振波谱仪 ;ＴＧＡ 7型热重分析仪 ;ＣＤＲ 1型差动热分析仪。1 2　Ｌ 磺基丙氨酸的电化学合成在自制Ｈ型电…     

Identification of degradation products formed during performic oxidation of peptides and proteins by high-performance liquid chromatography with matrix-assisted laser desorption/ionization and tandem mass spectrometry

Oxidation of proteins with performic acid is extensively used to cleave disulfide bonds. Due to its efficiency and many other advantages it deserves more attention especially in proteomics as a method for sample treatment. However, some unwanted degradations can occur during performic oxidation. In this work the degradation products during performic oxidation of two peptides and bovine serum albumin as model substrates were explored by coupling high-performance liquid chromatography (HPLC) to matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOFMS). In addition to well-known modifications such as oxidation of tryptophan and oxidation and chlorination of tyrosine, novel degradation products including nonspecific cleavage after asparagine or tryptophan, formylation of lysine, and beta-elimination of cysteine, were observed. Although almost all of these modification/degradation products except oxidation products of tryptophan were formed at sub-stoichiometric levels, they can cause confusion as a result of the sensitivity of mass spectrometry in analysis of the oxidized samples, especially in proteomics research. The results presented here will facilitate the interpretation of analytical data for performate-oxidized samples, and help to select appropriate methods for each unique sample.     

Oxidation of cysteine to cysteic acid in proteins by peroxyacids, as monitored by immobilized pH gradients.

It has often been debated whether the presence of persulfate in a polyacrylamide gel could lead to the oxidation of cysteine (Cys) in proteins to cysteic acid. In fact, direct incubation of bovine serum albumin (BSA) with peroxodisulfate and periodate barely alters the isoelectric point (pI) and does not produce any cysteic acid. In contrast, caroate (peroxomonosulfate) and perphthalate strongly lower the pI of BSA. In the former case it as demonstrated that 4-Cys (of a total of 35) were converted into cysteic acid. Perphthalate was found to be, by far, the strongest oxidant: 15 (of 35) Cys residues were oxidized to cysteic acid and all methionine groups were destroyed.     

Kinetics and mechanism of oxidation of <Emphasis Type="SmallCaps">l</Emphasis>-cystine by manganese(III) in sulfuric acid medium

The kinetics of oxidation of l-cystine by Mn^III have been studied in sulfuric acid medium at 30 °C. The reaction was followed spectrophotometrically at λmax = 500 nm. The reaction shows first order dependence on both [Mn^III] and [cystine]. It was found that the rate of the reaction decreases with increase of [H⁺] up to a certain point and then remains unchanged. The oxidation product of the reaction was found to be cysteic acid. A plausible mechanism has been proposed to account for the experimental results.     

Oxidation versus carboxamidomethylation of s-s bond in ranid frog peptides: Pro and contra for de novo MALDI-MS sequencing

Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 2674, 2636, 1874, and 1810 Da) were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b- and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions.