Peptide cysteic acid

Cysteic Acid Peptides from Hydrolyzate of Oxidized White Turkey Feather Calamus... 

Consden and Gordon (1950) have studied the cysteic acid peptides derived from the cystine peptides of wool as described on p. 40. After a preliminary group separation of the cysteic acid peptides by iono-phoresis, each fraction was fractionated on paper chromatograms with phenol and collidine. In Table VII are listed the peptides considered to be probably present and their approximate yield. The results were more clear-cut in this case than with the aspartic and glutamic acid... 

Cysteic Acid Peptides from Wool (Consden and Gordon, I960)... 

Cysteic Acid Peptides from Fraction B of Oxidized Insulin (Sanger and Tuppy, 1961a)... 

Figure 4-3. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (shaded) by per-formic acid (left) or reductive cleavage by 3-mercap-toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

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 ... 

L-Cysteic acid residues were produced within the peptide chain when submaxillary-gland glycoprotein from sheep was treated with alkali (pH 9.0), during 24 hours at room temperature in the presence of sulfite (0.1 M). The yield was —55% on the basis of the L-serine decomposed, and the corresponding derivative from L-threonine was not detected.167... 

To return now to chain A, Meedom (78) almost completely established the structure of this chain by the classic techniques of partial degradation. The single doubtful detail concerned the relative location of proline and alanine in the fourth and fifth positions. It is now known (67, 75) that aminopeptidase splits cysteic acid and glycine quickly and then valine much more slowly. This fact suggests that proline may be the fourth residue. When the DNP-derivative of the main peptide left behind by amino peptidase is submitted to partial degradation by acid, the dipeptide DNP. -Val.Pro is obtained. Thus the complete sequence may be written ... 

The relative ease of oxidation of a model disulfide, di-DNP-cystine, is illustrated in Table XIXe, where the amount of oxidant used was just in 50% excess (Ramacbandran, 1961). The yield of 73% DNP-cysteic acid is not far from values for yields of cysteic acid (80-90 %) from performic acid oxidation of cystine (Schram et ah, 1954). Proteins with reactive —SH, —S—S—, or free —NH2 groups therefore pose problems when side reactions in the oxidative cleavage of peptide bonds is to be avoided (cf. Section V, B). 

This mixture is subjected to electrophoresis in the perpendicular direction under the same conditions as those of the first electrophoresis. Peptides that were devoid of disulfides will have the same mobility as before, and consequently all will be located on a single diagonal line. In contrast, the newly formed peptides containing cysteic acid will usually migrate differently from their parent disulfide-linked peptides and hence will lie off the diagonal. These peptides can then be isolated and sequenced, and the location of the disulfide bond can be established. 

The combined ether extract (which may be washed with 0.01 N HCl to remove peptide material) is evaporated to dryness with a stream of nitrogen and 50 /il of 1 N HCl is added. After mixing, the thiazolinone derivative of the NH2 terminal residue is converted to the phenyl-thiohydantoin by incubation at 80°C (temperature block) for 10 min. If Asx or Glx residues are expected, 80°C for 3 min may give better results and for threonine, proline and serine, 50°C for 10 min may be better (Li and Yanofsky 1972). After cooling, the phenylthiohydantoin is extracted into ethyl acetate (100-200 /rl for each of 3 extractions), which is then evaporated with a stream of nitrogen. The phenylthio-hydantoins of arginine, histidine and cysteic acid will usually remain in the aqueous phase and may be recovered by lyophilization and dissolving the residue in methanol. 

Modification by performic acid oxidation Treatment of proteins with performic acid leads to the oxidation of cysteine and cystine residues to cysteic acid residues (Sanger 1949). Methionine residues are quantitatively converted to the sulfone (Hirs 1956), and tryptophan undergoes oxidative destruction (Toennies and Homiller 1942 Benassi et al. 1965). Other amino acids are not modified, provided that precautions are taken to avoid chlorination (Thompson 1954 Hirs 1956), or bromination (Sanger and Thompson 1963) of tyrosine residues. Cleavage of peptide bonds does not occur on performic acid oxidation at low temperature. 

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

W. Li, R.A. Boykins, P.S. Backlund, G. Wang, H.-C. Chen, Identification of pSerand pThr as cysteic acid and fi-methylcysteic acid residues in peptides by MS-MS sequencing. Anal. Chem., 74 (2002) 5701. 

Cleavage of disulfide bonds occurs before hydrolysis of the protein into peptides. Disulfide bonds may be cleaved oxidatively, or they may be reduced and alkylated. Treatment of the native protein with performic acid, a powerful oxidizing agent, breaks disulfide bonds and converts cystine residues to cysteic acid (Figure 3-11). Reduction of the disulfide linkage by thiols, such as d-mercaptoethanol, yields reactive sulfhydryl groups. These groups may be stabilized by alkylation with iodoacetate or ethyleneimine to yield the carboxymethyl or aminoethyl derivative, respectively. 

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