[21] Reduction and S-alkylation with acrylonitrile

Publisher Summary Alkylation of reduced proteins with acrylonitrile affords quantitative reaction with thiols. This chapter discusses the reduction and S-alkylation with acrylonitrile. This is because protein thiol groups formed by reduction of disulfide bonds with mercaptoethanol are effectively blocked by alkylation with acrylonitrile. The reaction is quantitative and the formation of cyanoethyl derivative is provided. The procedure of the recovery of S-cyanoethylated protein by lyophilization after dialysis is described. The acid hydrolysis and amino acid analysis is also focused. The chromatographic methods achieve separation and allow direct quantitation of S-carboxyethylcysteine. Under certain conditions of pH and length of exposure of reduced protein to aerylonitrile modification of lysine residues also occurs. The alkylation of amino groups of model compounds with acrylonitrile is a base-catalyzed nucleophilic addition, the rate of which is determined by the polar and steric characteristics of both reactants. The concentration of acrylonitrile may also be important in minimizing undesirable side reactions.