Cysteic acid, derivatizing

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

Fig. 7 Typical reversed-pbase separation of amino acids. Precolumn derivatization of a standard amino acid mixture was achieved employing FMOC. Resolution was achieved by gradient elution with acetonitrile, methanol, and acetate buffer (pH 4.2) on a C,8 column. Standard three-letter abbreviations for amino acids are used also, CySO H = cysteic acid. (From Ref. 164. Copyright 1983 Elsevier Science.)...

Fig. 8 Separation of standard mixture employing precolumn derivatization with AQC. Gradient elution with acetonitrile and acetate buffer (pH 5.0) was employed with a C18 column. Standard three-letter abbreviations for amino acids were used also, CA = cysteic acid, AMQ = hydrolyzed excess reagent, and nle = norleucine. Data was supplied by Stephen D. Smith, Ross Products Division of Abbott Laboratories, Columbus, OH.

The poor fluorescence of the cysteine, lysine, and ornithine derivatives may be a drawback of the technique. Cysteine yields weakly fluorescent properties due to its sulfydryl group (Cooper and Turnell, 1982). However, these sulfydryl groups can be blocked with iodoacetic acid, lodoacetamine, or acrylonitrile, with the result that fluorescent isoindoles can then be formed with OPA (Cooper and Turnell, 1982). Cysteine may also be oxidized to cysteic acid, which forms a highly fluorescent product with OPA. Following the oxidation of cysteine, however, it may be difficult to obtain high reproducibility with the OPA derivatization of amino acids, because the conditions for these two reac-... 

Fig. 2. The elution pattern of a standard mixture of OPA-derivatized primary amines, separated on a 5 (Jim Nucleosil C-18 column (200 X 4.6 mm id). The flow-rate was 1 mL/min employing the indicated gradient of metlianol and Na phosphate buffer (50 mA4, pH 5.25). Each peak represents 39 pmol except for those indicated below. 1, glutathione 2, cysteic acid 3, O-phosphoserine (19.5 pmol) 4, cysteine sulfinic acid 5, aspartic acid 6, asparagine (19.5 pmol) 7, glutamic acid 8, histidine 9, serine 10, glutamine 11, 3-methyl-histidine 12, a-aminoadipic acid (9.8 pmol) 13, citrulline (9.8 pmol) 14, carnosine 15, threonine,glycine 16, O-phosphoethanolamine 17, taurine (19.5 pmol) 18, p-alanine (19.5 pmol) 19, tyrosine 20, alanine 21, a-aminoisobutyric acid 22, aminoisobutyric acid 23, y-amino-ii-butyric acid 24, p-amino-u-butyric acid 25, a-amino-butyric acid 26, histamine 27, cystathione (19.5 pmol) 28, methionine 29, valine 30, phenylalanine 31, isoleucine 32, leucine 33, 5-hydroxytryptamine (5-H i ) 34, lysine. The chromatographic system consisted of a Varian LC 5000 chromatograph and a Schoeffel FS 970 fluorimeter.

In heat treated or stored food products several amino acids are not fully available because of derivatization or crosslinking reactions. Since 30 years furosine is known as a useful indicator of early Maillard reaction which is applied in food science, nutrition and medical biochemistry. Recently more sensitive analytical methods for furosine determination are available which have again increased the attractivity of this important indicator. Lately, N -carboxymethyllysine (CML) became available as another marker of special interest, because CML is a more useful indicator of the advanced heat damage by Maillard reaction than furosine. In addition, CML has the advantage to indicate reactions of lysine with ascorbic acid or ketoses such as fructose. Indicators for protein oxidation of sulfur amino acids are methionine sulfoxide and cysteic acid. An established marker for cross-linking reactions is lysinoalanine, which also indicates protein damages due to processing under alkaline conditions. Other markers formed as a consequence of alkaline treatment are D-amino acids. 

Precolumn derivatization with OPA has the same drawbacks as postcolumn derivatization. Secondary amino acids such as proline will not react unless first oxidized. Cysteine and cystine will not react, but can first be oxidized to cysteic acid. The addition of Brij or SDS to the reaction increases the fluorescence of lysine. 

Some nonprotein a-amino acids were derivatized with chiral variant of MR FDPA, according to modified Marfey s method [11]. The resulting diastereomers were separated by normal and reverse phase TLC in nanomole range. In normal phase TLC, the amino acids used were phenylglycine, cysteic acid, isovaline. 

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