CHEMICAL PROPERTIES 



339 



maleimides with thiolacetate and o-mercaptobenzoate. The addition product 

 of iV-ethylmaleimide and cysteine was first isolated by Smyth et al. (1960) 

 as the water-soluble >S-(iV-ethylsuccinimido)-L-cysteine. However, Fried- 

 mann (1952) pointed out than iV-ethylmaleimide and glutathione reacting 

 in very low concentration (0.004 mM) give various products depending 

 on the pH, the buffer, and other factors. The rate of reaction of iV-ethyl- 

 maleimide with thiols is very fast in most cases, e. g., with cysteine, glu- 

 tathione, or thiolacetate requiring less than a minute for completion near 

 neutrality (Fig. 3-1), the rate decreasing with decrease of the pH. Similar 



Fig. 3-1. Reaction of iV-ethylmaleimide 

 (1 mM) with glutathione (1 mM) at dif- 

 ferent pH's. (From Gregory, 1955.) 



results have been obtained by Roberts and Rouser (1958) (Fig. 3-2). It 

 may be noted that both the reaction with the SH groups and the spon- 

 taneous hydrolysis of iV-ethylmaleimide increase with rise in the pH; 

 however, around neutrality the rate of addition of thiols is so much faster 

 than the hydrolysis that the latter becomes relatively insignificant. 



iV-Ethylmaleimide has been applied to analyses for SH groups. Hanes 

 et al. (1951) and Gutcho and Laufer (1954) showed that the iV-ethylma- 

 leimide derivatives of thiols and cysteine peptides can be better separated 

 chromatographically than the parent compounds. Alexander (1958) pop- 

 ularized the spectrophotometric method for thiol assay, using the decrease 

 in the absorption at 300 m// as a measure of SH reaction, and this certainly 

 is the most convenient method, especially for kinetic studies. Concentra- 

 tions of thiol as low as 0.1 mM can be determined, and the method can 

 to some extent be used in tissue extracts. Benesch et al. (1956) observed 

 that a reddish color appears when the products of the reaction of iV-ethyl- 



