CHEMICAL PROPERTIES 293 



Reactions with Thiols 



Inasmuch as maleate is often considered to act as an SH reagent to in- 

 hibit certain enzymes, it is necessary to examine the reaction with thiols 

 in some detail. Morgan and Friedmann (1938 a) observed that cysteine, 

 glutathione, and thiolacetate lose their SH groups when incubated with 

 maleate at pH 7.4 and 37^, aerobically and anaerobically, whereas incuba- 

 tion with fumarate is ineffective. When maleate and thiolacetate are mixed 

 in equimolar concentrations (20 mM), half the SH groups disappear in 

 around 6 hr; as mentioned previously, fumarate is also formed and thus 

 the reaction with the SH groups does not go to completion. When maleate 

 is 80 mM and thiolacetate 20 mM, half reaction occurs in 80 min and 

 complete reaction in several hours. The reaction with glutathione is very 

 similar, but with cysteine the rate is greater and the reaction is more likely 

 to go to completion. The products of the reactions were isolated, in the case 

 of cysteine in 93.5% yield. The tricarboxylic ^-cysteinosuccinic acid was 

 crystallized and characterized by Morgan and Friedmann (1938 c). It was 

 pointed out that this substance might be found in hydrolyzates of proteins 

 treated with maleate, if reaction with protein cysteine occurs. The reaction 

 of maleate with thiolacetate was also investigated by Weller et al. (1957). 

 The addition of thiols to double bonds may involve a carbonium ion in- 

 termediate: 



and thus the rate will depend strongly on the pH, but this has not been in- 

 vestigated experimentally in the case of maleate. It may be noted that a 

 variety of related compounds react with thiols to varying degrees; these 

 include maleurate, iV-ethylmaleimide, and maleic hydrazide. 



Reactions with Proteins 



When rabbit muscle and liver protein preparations are incubated with 

 80 mM maleate at pH 7.4 and 37°, there is a gradual disappearance of the 

 SH groups, as determined by the nitroprusside reaction, and by 20 hr 

 no SH groups remain (Morgan and Friedmann, 1938 b). Denatured ovalbu- 

 min presumably reacts similarly. Unfortunately no kinetic data were 

 presented so the initial rates of reaction are unknown. Bovine seralbumin 

 forms complexes with various sulfonated azo dyes (e. g. methyl orange) 

 through electrostatic forces between the sulfonate groups and quaternary 

 N+ protein groups, with contributions from van der Waals' forces. Certain 

 organic acid anions are able to compete with the dyes for the positively 

 charged protein groups (Klotz, 1946). Maleate displaces methyl orange and 



