Reduction and carboxymethylation or carboxamidomethylation

Reduction and carboxymethylation (or carboxamidomethylation) The following generally applicable procedure is based on the method of Crestfield et al. (1963), and has been widely used with minor variations for the past ten years. 

Reduction with dithiothreitol (DTT). The use of DTT, in place of mercaptoethanol, has gained wide acceptance. The reduction proceeds essentially to completion at low levels of DTT, because of the thermodynamically favored formation of a 6-membered ring. The equilibrium constant at pH 7 and 25°C for the reduction of cystine by DTT is 10 , as compared to approximately 1 for mercaptoethanol (Konigsberg 1972). 

A representative procedure, patterned upon that employed for the complete reduction and alkylation of a yG immunoglobulin (Waxdal et al. 1968), is given below. 

In both of the above procedures, iodoacetamide may be substituted on an equimolar basis for iodoacetic acid with no alteration in reaction conditions. 

S-Carboxymethylcysteine is readily determined by amino acid analysis following acid hydrolysis under the usual conditions ( 2.5.3). The occurrence and extent of any alkylation of lysyl, histidyl, or methionyl residues would be revealed at the same time ( 2.5.10, 2.7.1, 2.8.1). 

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