CHEMICAL PKOPERTIES 13 



quite readily with glutathione at pH 9 where most of the SH groups are 

 dissociated, this being used as a basis for SH group determination by re- 

 cording of the H+ produced during the reaction. 



We may briefly turn to the question whether glutathione can be alkylated 

 by iodoacetate intracellularly. Schroeder et al. (1933 b) demonstrated that 

 appreciable amounts of glutathione in yeast can be reacted in the pH range 

 below 6. The reverse relationship to pH is observed in cellular work in that 

 inhibition and glutathione disappearance increase as the pH is decreased, 

 due to the greater penetration of the iodoacetate at the lower pH's (Fig. I- 

 14-21). Tsen and Collier (1960) have also shown that iodoacetate markedly 

 depletes erythrocytes of their glutathione, all being reacted after 90 min 

 exposure to 0.75 mM. Whatever the importance of glutathione and other 

 free thiols in cellular metabolism and function, it is evident that iodoacetate 

 can easily deplete them. 



Reactions with Amino Groups 



The emphasis on iodoacetate as an SH reagent has often obscured the fact 

 that reaction with amino groups can occur under certain conditions. Neu- 

 berg and Kobel (1931), in investigating the reason for the disappearance 

 of iodoacetate in yeast extracts, found that reaction with arginine, alanine, 

 and aniline occur, releasing iodide. The carboxymethylation of primary 

 amino groups occurs in two steps: 



R— NH2 + 1CH2C00" »- R-NH-CH2C00" + r + H^ 



CH2C00" 



R— NH-CH2COO" + ICH2COO" »- R-N + r + H^ 



VHsCOO" 



Both aliphatic and aromatic amines as well as ammonia are reacted. The 

 carboxymethylated products from glycine and alanine were isolated by Mi- 

 chaelis and Schubert (1934), but they pointed out that such reactions are 

 generally slower than with SH groups, so that it is possible to react the SH 

 group of cysteine before reaction with the amino group has occurred. At 

 elevated temperatures (80°) and in alkaline solutions the amino groups may 

 react quite rapidly, but at physiological pH and temperature it may require 

 a long time for significant alkylation (the half-reaction time for iodoacetate 

 and glycine is around 8 hr). 



The variation in reaction rate with pH is represented by a sigmoid curve 

 similar to the titration curve for glycine, indicating that it is the glycinate 

 anion HgN — CH.^ — COO" that reacts; the pK^ of the amino group is 9.6, 

 so that below a pH of 8 the rate is very slow (Brdicka, 1936). This was 



