338 3. A-ETHYLMALEIMIDE 



with thiols. Possibly some of the effects of 7V-ethylmaleimide on enzymes 

 and cells are unrelated to reaction with SH groups, and this compari- 

 son would be the most direct way of ascertaining the situation. One must 

 remember that iV-ethylmaleimide is a pyrrole derivative (iV-ethyl-2,5- 

 pyrroledione) and consequently could interfere with certain enzymes or 

 in metabolic pathways for this reason. 



Commercial iV-ethylmaleimide is apparently pure enough for most pur- 

 poses and what contaminants occur would not react with SH groups. 

 It may be recrystallized from ethanol. The compound is quite stable in 

 the solid state but in aqueous solution a hydrolytic ring opening leads to 



HC— C 



NH— CH2CH3 

 HC— coo" 



AT- E t hy 1 ma le a mate 



the formation of A'^-ethylmaleamate. This reaction occurs to a negligible 

 extent in the pH range below 7 but in alkaline sohitions the first order 

 hydrolysis rate increases linearly with the pH (Gregory, 1955). There 

 is a broad absorption band with a maximum near 300 m// for 7V-ethyl- 

 maleimide and the hydrolysis can be followed by a decrease in absorption 

 at this wavelength. The percentage of hydrolysis in 6 hr is roughly as 

 follows: pH 7—10%, pH 8—60%, and pH 9—90%. Alkaline solutions of 

 iV-ethylmaleimide may be sufficiently stable for short experiments but 

 fresh solutions should be made for each experiment. Reduction of the 

 C=C bond to form the succinimide does not occur readily — the polaro- 

 graphic half-wave potentials for several A'^-substituted maleimides lie near 

 — 0.8 V (Muir, 1959) — but in biological systems it should not be neglected, 

 although nothing is known of enzyme systems catalyzing this reaction. 

 I have not been able to find the solubility of A-ethylmaleimide, but so- 

 lutions up to 200 mM can be prepared. 



Reaction with Thiols 



The unsaturated imides react rapidly with thiols under physiological 

 conditions, yielding products of the following type: 



R'— S O 



\ // 

 HC— C 



H,C-C 

 O 



as described by Marrian (1949) for the reactions of A-ethyl- and A- phenyl- 



