Cysteine 5-carboxymethyl derivative

The thiohydantoin-amino acid standard HPLC chromatogram (Figure 1) shows the elution times for each of the 20 common amino acid derivatives (approximately 50 pmols) including thiohydantoin-Pro (P) and the common peak designated S/C, identifying Ser and Cys residues. The relative retention time for the S-carboxymethyl derivative of cysteine is indicated by the arrow. 

As already pointed out, the chief advantage of using derivatives of haloacetates as modification reagents is that the carboxymethyl derivatives of most of the nucleophilic amino acids have been described. The chromatographic behavior of the carboxymethylated derivatives of lysine, cysteine and histidine have been described in ch. 2. O-Carboxymethyl tyrosine appears as a single peak in the long column... 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

EXAMPLE 3.29 lodoacetate forms an S-carboxymethyl derivative of cysteine residues, thus blocking the —SH group and rendering it unavailable for disulfide bond formation. When it modifies the —SH group in the active site of an enzyme, it inhibits that enzyme. 

Cyst(e)ine occurs in biological tissues and fluids, both in the free sulfhydryl form, cysteine, and in the disulfide form, cystine In the methods of sample preparation described above, cysteine is oxidized to cystine, and the proportion of the two forms in a sample cannot be determined. A method was devised to accomplish this, involving immediate addition to the sample of iodoacetate, which rapidly reacts with cysteine, converting it quantitatively to the stable S-carboxymethyl derivative (Brigham et al., 1960). Cystine is unaffected by iodoacetate. Both cystine and S-carboxymethylcysteine can be quantified by amino acid analysis. This same strategy can be used with other sulfhydryl compounds, such as homocyst(e)ine. 

Recent studies on separation optimization showed that accurate control of mobile phase pH was essential to successfully resolve a number of important non-hydrolysate amino acids. With good control of a complex gradient profile the system could resolve a mixture of amino acids including, Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids (4). However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method between laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. The current study demonstrates the utility of quaternary HPLC gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. 

Inactivation of alcohol dehydrogenase from yeast with 14C-labeled [3-(3-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation showed the presence of 1-carboxymethyl histidine66. After inactivation of the enzyme with labeled [3-(4-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation, S-carboxymethyl cysteine was identified in the protein. In the case of glyceraldehyde-3-phosphate dehydrogenase, treatment with either coenzyme analogue leads to the modification of the cysteine residue. Treatment with [14C]nicotinamide-5-bromo-4-methylimidazole dinucleotide did not reveal any modified amino-acid-residues. The labeled nicotinamide residue split off during the recovery of the inactivated enzyme. Attempts to synthesize an inactivator labeled with a 14C-acetyl residue did not give satisfactory yields. If the enzyme-coenzyme derivative was treated with tritiated sodium boron hydride, tritium could be introduced (Fig. 22). Studies with... 

The protein is completely hydrolyzed by acid (6 N HCl, 24 hours or longer at 110°C, under vacuum or inert gas) to its constituent amino acids and the resultant hydrolysate is evaporated to dryness. The amino acid composition is determined on protein hydrolysates obtained after 24,48, and 72 hours of acid treatment. The content of amino acids with bulky aliphatic side chains such as isoleucine, leucine, and valine, which undergo slow hydrolysis, is calculated from an extrapolation of the hydrolysate data to infinite time. The content of hydroxyl-containing amino acids, which are slowly destroyed during hydrolysis, is obtained by a corresponding extrapolation to zero time. Since cysteine, cystine, and methionine residues are somewhat unstable to hydrolysis, these residues are oxidized to cysteic acid and methionine sulfone, respectively, with performic acid before quantitative analysis. Cysteine, or half-cystine, is quantitated as a derivative such as carboxymethyl cysteine after reduction and alkylation, a necessary prerequisite to subsequent sequence analysis. Tryptophan... 

In the case of amino acid analysis, the quantification of cysteine can be difficult because it is oxidized to cystine during acid hydrolysis. To circumvent this problem, cysteine can be oxidized to cysteic acid with performic acid prior to analysis. Alternatively, cysteine can be converted to the pyridyl ethyl derivative and subsequently detected by postcolumn reaction with ninhydrin. Still another method involves the production of carboxymethyl cysteine following alkylation with iodoacetic acid. All of these cysteine derivatives can be separated by either reversed-phase precolumn or ion-exchange postcoT umn methods. 

Mucoadhesive polymers, such as derivatives of PAA and thiol-functionaUzed polymers, enable a strong attachment to cysteine groups of glycoproteins from mucus layer, resulting in an increased residence time and enhanced permeability [61, 62]. Functionalization with thiol groups notably inaeases the mucoadhe-sion strength of chitosan, PAA derivatives, alginate or carboxymethyl cellulose [63-65]. 

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