Dehydroalanine analysis

Fig. 25-15). In every case it is NH3 or an amine, rather than an OH group, that is eliminated. However, the mechanisms probably resemble that of fumarate hydratase. Sequence analysis indicated that all of these enzymes belong to a single fumarase-aspartase family.64 65 The three-dimensional structure of aspartate ammonia-lyase resembles that of fumarate hydratase, but the catalytic site lacks the essential HI 88 of fumarate hydratase. However, the pKa values deduced from the pH dependence of Vmax are similar to those for fumarase.64 3-Methylaspartate lyase catalyzes the same kind of reaction to produce ammonia plus czs-mesaconate.63 Its sequence is not related to that of fumarase and it may contain a dehydroalanine residue (Chapter 14).66... 

Dehydroalanine 116 desmosine 48, 49 diazo compounds 157 aryl diazonium salts, reactive properties 157 synthesis 160 diazoacetates, analysis of products 165 reactive properties 162 synthesis 164 diazoketones, analysis of products 162 conversion to haloketones 139 reactive properties 165 synthesis 140 diazomethane preparation 141 reactive properties 162 diazonium salts 89 diazonium-IH-tetrazole 90, 95 3,4-dihydroxyproline 52, 53 diimidoesters 69 diisopropylfluorophosphate 130 2,3-dimethylmaleic anhydride 83 dinitrophenylation 79 disulfide bond reduction 103... 

Similar to the analysis of ordinary proteins, phosphoproteins also need to be cleaved into small manageable peptide fragments any one of the proteases listed in Table 1 can be used. The concept of chemical tagging through /3-elimination/Michael addition reactions has been adopted to facilitate the cleavage process. The [3-elimination reaction converts phosphoserine and phosphothreonine to dehydroalanine and... 

When nisin, fragments of nisin (still containing dehydroalanine and lysine), and subtilin were treated under basic conditions, the formation of lysinoalanine was observed in each case. Treatment, for instance, of the carboxyl-terminal fragment of nisin (Figure 11) under these conditions (11) (IN N-ethylmorpholine, pH 10.65, 7 days, room temperature) followed by total hydrolysis and amino acid analysis showed the presence... 

One problem of prime importance is the reliable determination of the number of residues of ,/3-unsaturated amino acids in proteins. Direct amino acid analysis subsequent to total hydrolysis of proteins is not feasible. The ,/3-unsaturated amino acids are subject to degradation with the formation of amide (ammonia) and -keto-acid. The numbers and types of ,/3-unsaturated amino acids in nisin (1) and subtilin (10) and in the fragments of the two peptides were, nevertheless, determined by amino acid analysis, only, however, after the addition of mercaptan across the double bonds of dehydroalanine and dehydrobutyrine (19). Using benzylmercaptan, the addition products are S-benzylcysteine (from dehydroalanine) and /3-methyl-S-benzylcysteine (from dehydrobutyrine). The two thioether amino acids are eluted from ion exchange columns of the amino acid analyzer free from interference by other amino acids... 

We are aware of no reports finding dehydroalanine in food proteins. During acid hydrolysis the dehydroalanine is broken down to ammonia and pyruvic acid therefore, only the ammonia would be detected in routine amino acid analysis. In summarizing the many reactions of dehydroalanine in proteins, Friedman (25) reported that 14 crossiinked amino acids can be formed in proteins. If one considers the isomeric forms that could exist, the number expands to at least 53 derivatives. 

Figure 1 summarizes the potential pathways involved in the formation of dehydroalanine. It appears that dehydroalanine can be formed in a variety of amino acids protein, suggesting that any or all of the routes in Figure 1 could be involved in dehydroalanine formation. Table 1 contains results of partial amino acid analysis of several alkaline treated proteins. The results support the suggestion that both serine and cystine or their derivatives can be sources of dehydroalanine and subsequently the lysinoalanine measured in the proteins. In casein there is substantial LAL formation with a measurable loss in serine. In isolated soy protein and lactalbumin it can be seen that cystine shows the most significant losses. It should be noted that a significant portion of the serine in casein is present as phosphoserine. The relatively rapid 6-elimination of phosphoserine (15) accounts for the formation of considerable quantities of dehydroalanine and subsequently the substantial levels of LAL found in casein. In addition, as mentioned above, the presence of calcium would accelerate dehydroalanine formation from the phosphoserine present in the casein. The variability of... 

The reaction of lysine with dehydroalanine was studied by Snow et al. (21) using the N-a-acetyl-dehydroalanine methyl ester as a model. The studies with the model compound are difficult to compare with reactions that occur in proteins because proteins are such complex molecules and analysis is difficult. The comparison of the variety of proteins which form LAL suggests that although dehydroalanine may be formed by a variety of pathways, the Michael addition between lysine and dehydroalanine is rapid, particularly at higher pHs. 

Although the results are consistent with a B-elimination reaction leading to formation of dehydroalanine, the conclusions are based on the assumption that the absorbance at 241 nm is associated with dehydroalanine side chains derived from cystine residues. This assumption may not always be justified for the following reasons. First, alkali treatment of casein which has very few or no disulfide bonds also yields significant amounts of dehydroalanine residues (52). These presumably arise from serine side chains. Second, Nashef et al. (41) cite evidence that other functionalities may contribute to the 241 nm absorption. These considerations suggest that there is a need to directly measure dehydroalanine in proteins. This is now possible with our method (52), whereby the dehydroalanine residues are first transformed to S-pyridylethyl side chains by reaction with 2-mercaptopyridine (Figure 12). Amino acid analysis of the acid-hydrolyzed protein permits estimation of the dehydroalanine content as S-B-(2-pyridylethyl)-L-cysteine along with the other amino acids. 

In a related study, Masri and Friedman (1982) developed a procedure for detecting dehydroalanine in alkali-treated proteins based based on addition of the SH group of 2-mercaptoethylpyridine to the double bond of dehydroalanine to form S-p-(2-pyridylethyl) cysteine. The cysteine derivative can be assayed, after acid protein hydrolysis, by standard amino acid analysis techniques (Friedman et al., 1979). 

The side chains of proteins can undergo post-translational modification in the course of thermal processes. The reaction can also result in the formation of protein cross-links. A known reaction which mainly proceeds in the absence of carbohydrates, for example, is the formation of dehydroalanine from serine, cysteine or serine phosphate by the elimination of H2O, H2S or phosphate. The dehydroalanine can then lead to protein cross-links with the nucleophilic side chains of lysine or cysteine (cf. 1.4.4.11). In the presence of carbohydrates or their degradation products, especially the side chains of lysine and arginine are subject to modification, which is accompanied by a reduction in the nutritional value of the proteins. The structures of important lysine modifications are summarized in Formula 4.95. The best known compounds are the Amadori product -fructoselysine and furosine, which can be formed from the former compound via the intermediate 4-deoxyosone (Formula 4.96). To detect of the extent of heat treatment, e. g., in the case of heat treated milk products, furosine is released by acid hydrolysis of the proteins and quantitatively determined by amino acid analysis. In this process, all the intermediates which lead to furosine are degraded and an unknown portion of already existing furosine is destroyed. Therefore, the hydrolysis must occur under standardized conditions or preferably by using enzymes. Examples showing the concentrations of furosine in food are presented in Table 4.13. 

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