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.     

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.     

RPHPLC determination of L- and D-cystine and cysteine as cysteic acid

Summary In the quantitative analysis of cystine and cysteine, before hydrolysis the compounds are often oxidized to cysteic acid with performic acid. The applicability of this process to the quantification of the enantiomers of cystine and cysteine has been examined. An RPHPLC analytical method was developed for determination of the amount of cysteic acid enantiomers and the rate of conversion during oxidation from cystine and cysteine into cysteic acid was determined. Racemization of L-cysteine was not significant during oxidation with performic acid and the process can, therefore, be applied before hydrolysis during quantification of enantiomers of these compounds. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

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。     

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]。     

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.     

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.     

Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]⁻ ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C_oxLRRASLG) [M – H + O₃]⁻ ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d _i fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a _i ions followed by the elimination of the side chain to form d _i ions).     

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.     

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…     

Cystine and cysteine analysis as the same phenylthiocarbamyl derivatives by HPLC in protein hydrolyzates

Summary A new approach is described, and a novel explanation presented, for the high performance liquid chromatographic analysis of cystine and cysteine as their phenylthiocarbamyl derivatives. PTC cystine and cysteine have been eluted with the same retention times and molar responses, most probably due to electrophilic attack of phenylisothiocyanate on cystine resulting in the scission of the disulfide bond yielding two moles of cysteine. Further, total PTC cystine and cysteine have been measured both in model solutions and in standard protein hydrolyzates (lysozyme, bovine albumin, ribonuclease) with the same linearity as the other ineteen amino acids. The reproducibility of the measurements, at the 250–750 pmole level, proved to be 4.1% (Relative Standard Deviation %) or less.     

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.     

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.     

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.     

Advantages of derivatization by osmium tetroxide and 2,2'-bipyridine for matrix-assisted laser desorption/ionization post-source decay fragment ion analysis of peptides

Matrix-assisted laser desorption/ionization--post-source decay (MALDI-PSD) fragment ion analysis is frequently used for peptide sequence determination. PSD fragmentation is often changed or improved in terms of, e.g., sequence coverage, after derivatization. In this work, the influence of modification by an osmium tetroxide-bipyridine reagent (Os,bipy) on the MALDI-PSD behaviour of peptides is studied. The reagent modifies peptides specifically at tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid. As a result the masses of some of the fragments are specifically shifted in case of peptides containing a methionine by +32 Da and, in cases of peptides containing a cysteine residue, by +48 Da. In addition, due to the change in protonation properties of a peptide after oxidation, fragments containing cysteic acid are in most cases totally suppressed. This effect significantly facilitates peptide sequence determination. Improvement of MALDI-TOFMS and PSD analysis after the reaction with Os,bipy is demonstrated for examples involving derivatives of humanin, a novel neuroprotective peptide.     

Electrocatalytic oxidation and flow-injection determination of sulfur amino acids on a glassy carbon electrode modified by a nickel(II) polytetrasulfophthalocyanine film

The electrochemical oxidation of sulfur amino acids, i.e., cysteine, cystine, and methionine, is studied on a glassy carbon electrode modified by a film of nickel(II) polytetrasulfophthalocyanine (poly-NiTsPc). Poly-NiTsPc demonstrates a selective mediator activity in the oxidation of sulfur amino acids, depending on the pH of solution. The proper conditions for fabricating a polymer film on the surface of glassy carbon are found and the conditions of registering the maximal electrocatalytic effect on the modified electrode are determined. A procedure is proposed for the voltammetric determination and amperometric detection of cysteine, cystine, and methionine on an electrode coated by a poly-NiTsPc film under the conditions of flow-injection analysis (FIA). The linear relation of the electrocatalytic response of a composite electrode to amino acid concentration is observed to the level n × 10^?6 M in the static mode and n × 10^?9 M under FIA conditions.     

Determination of Calcium and Copper in Feedstuffs by Atomic Absorption Spectrometry Following a Digestion Procedure with H2SO4 + H2O2

Feedstuffs can be effectively dissolved by treatment with concentrated sulfuric acid and 30% hydrogen peroxide in approximately 30 min for the rapid determination of elements at both the 1% level and the trace level by flame and electrothermal atomic absorption spectrometry, respectively. This paper reports a new determinative method for calcium (10 g · kg⁻¹) and copper (10 mg · kg⁻¹) based on this procedure. The relative standard deviation of the results was typically ca. 5%.     

Spectrophotometric determination of Si(IV) with 3,6-dichloro-2,5-dihydroxy-1,4-benzoquinone

A new rapid spectrophotometric method for the determination of Si is described. Chloranilic acid (CA) reacts with Si(IV) forming a complex, which is stable for several hours. The procedure is suitable for the determination of Si in the concentration range of 0.5–5.0 μmol/ml, at pH 1.25 ± 0.05 measured at 370 nm. The relative standard deviation at the level of 1.0 μmol/ml Si is ± 3.5%. Of the foreign ions investigated, Fe, Ti, Mo, and PO₄³⁻ interfere significantly.     

Negative Ion Fragmentation of Cysteic Acid Containing Peptides: Cysteic Acid as a Fixed Negative Charge

We present here a study of the collision induced dissociation (CID) of deprotonated cysteic acid containing peptides produced by MALDI. The effect of cysteic acid (C_ox) position is interrogated by considering the positional isomers, C_oxLVINVLSQG, LVINVLSQGC_ox, and LVINVC_oxLSQG. Although considerable variation between the CID spectra is observed, the mechanistic picture that emerges involves charge retention at the deprotonated cysteic acid side chain. Fragmentation occurs in the proximity of the cysteic acid group by charge directed mechanisms as well as remote from this group to form ions, which may be rationalized by charge remote mechanisms. Additionally, the formation of the SO₃^–• ion is observed in all cases. Fragmentation of C_oxLVINVLSQC_ox provides both N- and C-terminal, y and b ions, respectively indicating that the negative charge may be retained at either of the cysteic acids; however, there is some evidence that charge retention at the C-terminal cysteic acid may be preferred. Fragmentation of tryptic type peptides containing a C-terminal arginine or lysine residue is considered through comparison of three peptides C_oxLVINKLSQG, C_oxLVINVLSQK, and C_oxLVINVLSQR. Lastly, we rationalize the formation of b _n–1 + H₂O and a _n–1 ions through a mechanism involving rearrangement of the C-terminal residue to form a mixed anhydride intermediate.