Cystine in proteins

L Sottrup-Jensen. Determination of half cystine in proteins as cysteine from reducing hydrolysates. Biochem Mol Biol Int 30 789-794, 1993. 

V Barkhold, AL Jensen. Amino acid analysis determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal Biochem 177 318-322, 1989. 

Cysteic acid is the major product of performic acid oxidation of cysteine and cystine in proteins, and is usually produced in yields of more than 90%. Cysteic acid is not retarded by the resins generally used in amino acid analyzers and therefore elutes at the breakthrough volume (about 42% of the elution volume of aspartic acid). However, since other substances such as O-phosphoserine can also elute in this position, it is essential to analyze hydrolysates made both before and... 

The thiol group of Cys is the most reactive side residue. The thiolate anion is a potent nucleophile and the thiol is a week acid with pKj = 8.37. Cys serves as the active site residues of many oxidoreductases. Cys residues form complexes of varying stability with a variety of metal ions. It reacts with organic mercurials stoichiometiically. Thiol residues of Cys cross link to form disulfide bonds (cystine) in proteins. Thiols and disulfides undergo rapid exchange and redox reactions. 

Photochemistry of proteins is derived from diose of its amino acid residues. In addition, important photochemical processes in proteins involve the splitting of the disulfide bridges. Furthermore, DNA-protein cross-linking involves the addition of thymine in DNA with cystine in protein. This addition process has been discussed above. In the case of DNA, UV irradiation can also lead to the breaking of either one or both DNA strand and infra- and inter-molecular DNA cross-linking. The photochemistry of RNA is very similar to that of DNA. 

Sulfur. Sulfur is present in every cell in the body, primarily in proteins containing the amino acids methionine, cystine, and cysteine. Inorganic sulfates and sulfides occur in small amounts relative to total body sulfur, but the compounds that contain them are important to metaboHsm (45,46). Sulfur intake is thought to be adequate if protein intake is adequate and sulfur deficiency has not been reported. Common food sources rich in sulfur are Hsted in Table 6. 

Protein Hydrolysis. Acid hydrolysis of protein by 6 MHQ in a sealed tube is generally used (110°C, 24-h). During hydrolysis, slight decomposition takes place in serine (ca 10%) and threonine (ca 5%). Cystine and tryptophan in protein cannot be deterruined by this method because of complete decomposition. 

Cysteine [52-90 ] is a thiol-bearing amino acid which is readily isolated from the hydrolysis of protein. There ate only small amounts of cysteine and its disulfide, cystine, in living tissue (7). Glutathione [70-18-8] contains a mercaptomethyl group, HSCH2, and is a commonly found tripeptide in plants and animals. Coenzyme A [85-61-0] is another naturally occurring thiol that plays a central role in the synthesis and degradation of fatty acids. 

One of the amino adds commonly found in protein hydrolysates is called cystine it has the following structure ... 

D. B. Volkin and A. M. Klibanov, Thermal destruction processes in proteins involving cystine residues, J. Biol. Chem, 262, 2945 (1987). 

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

Schneider JF, Westley J. 1969. Metabolic interrelations of sulfur in proteins, thiosulfate, and cystine. J Biol Chem 244 5735-5744. 

Now let us examine the distribution and position of disulfides in proteins. The simplest consideration is distribution in the sequence (see Fig. 51), which is apparently quite random, except that there must be at least two residues in between connected half-cystines. Even rather conspicuous patterns such as two consecutive halfcystines in separate disulfides turn out, when the distribution is plotted for the solved structures (Fig. 51), to occur at only about the random expected frequency. The sequence distribution of halfcystines is influenced by the statistics of close contacts in the three-dimensional structures, but apparently there are no strong preferences of the cystines that could influence the three-dimensional structure. 

There is a correlation between the backbone conformations which commonly flank disulfides and the frequency with which disulfides occur in the different types of overall protein structure (see Section III,A for explanation of structure types), although it is unclear which preference is the cause and which the effect. There are very few disulfides in the antiparallel helical bundle proteins and none in proteins based on pure parallel /3 sheet (except for active-site disulfides such as in glutathione reductase). Antiparallel /3 sheet, mixed /8 sheet, and the miscellaneous a proteins have a half-cystine content of 0-5%. Small proteins with low secondary-structure content often have up to 15-20% half-cystine. Figure 52 shows the structure of insulin, one of the small proteins in which disulfides appear to play a major role in the organization and stability of the overall structure. 

The formation of bromophenylmercapturic acid from bromobenzene and cystine in the organism, if it had the formula given it by Baumann now seemed scarcely possible, unless an isomeric a-thio-y8-aminopropionic acid were also present in the protein molecule together with the di-/8-thio-a-aminopropionic acid or cystine. The investigation of their constitution was therefore taken up by Friedmann in 1904, who succeeded in showing that they were also derived from -thio-a-aminopropionic acid and not from the isomeric a-thio-/8-aminopropionic acid. 

Fischer and Suzuki soon afterwards showed that Neuberg and Mayer s stone cystine contained tyrosine, and that its different behaviour to protein cystine was due to the presence of this compound. Rothera also could find no difference between stone cystine and protein cystine, and further, Gabriel s synthesis of isocysteine or a-thio-/8-aminopropionic acid and isocystine, which had quite different properties to cystine, though the two were much alike in many of their reactions, proved that stone cystine and protein cystine must be identical substances. Finally, it has been shown by Friedmann that a-thiolactic acid, ammonia PT. I. 4... 

Casein is low in sulphur (0.8%) while the whey proteins are relatively rich (1.7%). Differences in sulphur content become more apparent if one considers the levels of individual sulphur-containing amino acids. The sulphur of casein is present mainly in methionine, with low concentrations of cysteine and cystine in fact the principal caseins contain only methionine. The whey proteins contain significant amounts of both cysteine and cystine in addition to methionine and these amino acids are responsible, in part, for many of the changes which occur in milk on heating, e.g. cooked flavour, increased rennet coagulation time (due to interaction between /Mactoglobulin and K-casein) and improved heat stability of milk pre-heated prior to sterilization. 

Asparagine and glutamine are the amides of two other amino acids also found in proteins, aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules or residues are joined by a disulfide bond (Fig. 3-7). The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains. 

FIGURE 3-26 Breaking disulfide bonds in proteins. Two common methods are illustrated. Oxidation of a cystine residue with performic acid produces two cysteic acid residues. Reduction by dithiothreitol to form Cys residues must be followed by further modification of the reactive —SH groups to prevent re-formation of the disulfide bond. Acetylation by iodoacetate serves this purpose. 

Disulfide bond The side chain of cysteine contains a sulfhydryl group (-SH), which is an important component of the active site of many enzymes. In proteins, the -SH groups of two cysteines can become oxidized to form a dimer, cystine, which contains a covalent cross-link called a disulfide bond (-S-S-). (See p. 19 for a further discussion of disulfide bond formation.)... 

Whey proteins are slightly superior to casein because of the limiting quantity of the total sulfur-containing amino acids (methionine plus cystine) in casein. However, because whey proteins have a relative surplus of these amino acids, casein and whey proteins, as found in milk,... 

Figure 3-14 shows the spectra of N-acetyl ethyl esters of all three of the aromatic amino acids and of cystine. To a first approximation, the absorption spectra of proteins can be regarded as a summation of the spectra of the component amino acids. However, the absorption bands of some residues, particularly of tyrosine and tryptophan, are shifted to longer wavelengths than those of the reference compounds in water. This is presumably a result of being located within nonpolar regions of the protein. Notice that the spectra for tyrosine, phenylalanine, and cystine in Fig. 

The reaction that leads to BCA color formation as a result of the reduction of Cu2+ is also strongly influenced by the presence of any of four amino acid residues (tyrosine, tryptophan, cysteine, or cystine) in the amino acid sequence of the protein. Unlike the Coomassie dye-binding (Bradford) methods, which require a minimum mass of protein to be present for the dye to bi nd, the presence of only a single amino acid residue in the sample may result in the formation of a colored BC A-Cu+ chelate. This is true for any of the four amino acids cited above. Studies done with di- and tripeptides indicate that the total amount of color produced is greater than can be accounted for by the simple addition of the color produced with each BCA-reactive amino acid, so the peptide backbone must contribute to the reduction of copper as well. 

Determination of protein concentration by measuri ng absorbance at 280 nm (A2g0) is based on the absorbance of UV light by the aromatic amino acids tryptophan and tyrosine, and by cystine, disulfide bonded cysteine residues, in protein solutions. The measured absorbance of a protein sample solution is used to calculate the concentration either from its published absorptivity at 280 nm (a280) or by comparison with a calibration curve prepared from measurements with standard protein solutions. This assay can be used to quantitate solutions with protein concentrations of 20 to 3000 pg/ml. 

Our data also indicate greater stability of the amino acids in beef than was observed in the case of 7-ray irradiation of insulin. In the latter case, Drake et al. (8) found that in addition to cystine, tyrosine, phenylalanine, proline, and histidine were quite radio sensitive. Instability of tyrosine in proteins on 7-ray irradiation has also been reported by Hatano (16), who states that in protein, tyrosine is the most sensitive amino acid. Kolo-miichenko and Morozova (24) found about 20% destruction of tryptophan, tyrosine, and histidine following 1.5 Mrads 7-ray irradiation of egg albumin. 

---