Cysteine iodoacetic acid reaction

Problem 26.10 1 Show the structure of the product you would expect to obtain by SN2 reaction of a cysteine residue with iodoacetic acid. 

Bradbury and Smyth (1973) advocate the use of 3-bromopropionic acid in the place of iodoacetic acid, or its amide, in the alkylation reaction. S-carboxyethylcysteine was found to be completely stable at acid pH, and could be readily quantitated by amino acid analysis ( 2.5.3). The half-life of the reaction of the SH-group of cysteine (2 jumoles/ml) with 3-bromopropionate (200 pmoles/ml) at pH 8 and 30°C was approximately 10 min. Under the same conditions, the reaction of cysteine and iodoacetate was found to be virtually instantaneous (Bradbury and Smyth 1973). 

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

Write the mechanism for the reaction of a cysteine residue with iodoacetic acid. 

The experimenter separates heterooligomers before cleaving them into subunits. Then she denatures the purified subunit and reduces possible disulfide bridges. A carboxymethylation with iodoacetic acid protects against the reoxidation of the cysteines (Lind and Baker 1982). This reaction requires a sure instinct, in that other amino acids such as methionine, lysine, and histidine also react at high concentrations of iodine acetate or at the wrong pH. If you just need a few partial sequences, you can skip the carboxymethylation. 

To determine the number of disulfide bonds, the protein is reduced and alkylated as described in Section 8.5.1 by reacting it with either dithiothreitol or 2-mercaptoethanol, followed by reaction with iodoacetic acid. The molecular masses of the native protein (Mnat) and reduced and alkylated protein (Mr+a) are determined with MALDl-MS or ESl-MS. S-Carboxymethylation increases the mass of each cysteine residue by 59 Da. From the change in the molecular mass, the number of cysteine residues (Ncys) and hence the number of disulfide bonds (Ns-s) can be estimated using... 

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. 

Specific modification of Cys-46. Li and Vallee 86,87) and Harris 86) found that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate. The modified enzyme is inactivated and this cysteine residue, Cys-46 92), was suggested to be at the active site of the enzyme. The same residue in the S subunit is also especially reactive 20,94). The modification is preceded by anion binding of the iodoacetate and stimulated by the presence of imidazole 140,142,142). By using these facts and working with the crystalline enzyme, it is possible to achieve a highly specific and complete modification (ISO). X-ray studies of the carboxymethylated enzyme and the reaction mechanism of this modification are described in Section II,H. The carboxymethylation has been used to establish that both the EE 19) and SS 20) isozymes are active in u oxidations of fatty acids. 

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