340 



3. iV-ETHYLMALEIMIDE 



maleimide and homocysteine are made somewhat alkaline, and then found 

 that a number of thiols produce this color, including glutathione and 

 lipoate. This test is rather sensitive, detecting as little as 0.01 m.M thiol, 

 and can be used to visualize thiol spots on chromatographic paper, but 

 there is still some doubt as to its complete specificity (see below). Seligman 



Fro. 3-2. Reactions of iV-ethylmaleimide (1 mM) 



with cysteine (1 mM) and seralbumin at pH 6 



and 25° determined by absorption changes at 300 



m/i. (From Roberts and Rouser, 1958.) 



(1954) developed a chromogenic derivative of iV-ethylmaleimide for de- 

 tecting thiols histologically, this being iV-(4-hydroxy-l-napthtyl)malei- 

 mide, which reacts much faster than 2,2'-dihydroxy-6,6'-dinaphthyldisul- 

 fide (DDD). Tuppy (1959) mentions the yellow iV-(4-dimethylamino-3,5- 

 dinitrophenyl)maleimide, which has the advantage that yellow peptide 

 derivatives can be isolated from proteins treated with it. More recently, 

 Clark- Walker and Robinson (1961) have used iV-2,4-dinitroanilinomaleimide 

 as a colored reagent and have shown that it can be applied to the labeling 

 of protein SH groups. 



The initial addition products of maleimides with thiols sometimes un- 

 dergo further interesting reactions. The red color observed by Benesch 

 et al. (1956) may be due to polymerization of A^-ethylmaleimide in some 

 cases, Smyth et al. (1960) having found a pink crystalline precipitate 

 following the reaction of iV-ethylmaleimide and cysteine at pH 7.4, just 

 as in the reaction of A^-ethylmaleimide with imidazole. The cysteine which 

 is reacted with the iV-ethylmaleimide probably undergoes intramolecular 



