14 1. lODOACETATE AND lODOACETAMIDE 



related to the unbonded electron pair possessed by the NHg group, the 

 reaction possibly proceeding through an intermediate: 



'OOC— CH2— NH2 + I-CH2— coo" >- "OOC-CHa-N^— CH2COO" 



H 



"OOC-CH2— NH— CHzCOO" 



Even at pH 13, glycine at 33 mM, and iodoacetate at 10 mM, it requires 

 3 hr for half-reaction. Schubert (1936) pointed out, however, that various 

 amino groups react at quite different rates, and particularly that tertiary 

 amines react faster than primary or secondary amines. Reactions with py- 

 ridine, nicotinate, and nicotinamide are fairly rapid; this may in part be 

 related to the lower pZ^'s of these substances (e.g., piiC^ is 5.21 for pyridine) 

 so that a greater fraction is present in the dissociated form. He also pointed 

 out that iodoacetate generally reacts more rapidly than iodoacetamide with 

 amino groups (Table 1-2), in contrast to the situation with SH groups. 

 Again it must be emphasized that even the fastest reacting amines give 

 rates only around 1/400 that for glutathione or cysteine. Histidine reacts 

 slowly with iodoacetate and bromoacetate, presumably in the following way; 



CHaCOO" 



HN— CH N-CH 



/I - / I - + 



— CH2— C^ I + XCH2COO * CHa-C^ I 4- X + H 



HC— N HC— N 



Despite the slowness of the reaction with free histidine, one histidine residue 

 in ribonuclease reacts quite rapidly (Barnard and Stein, 1959). The studies 

 with amino acids and other simple amines indicate that reactions of iodo- 

 acetate with amino groups must be unimportant relative to reactions with 

 SH groups, but we shall see that within the structure of certain proteins 

 the amino groups may be in a special state of reactivity, so that in work 

 with enzymes the amino groups cannot always be ignored. In addition, one 

 must bear in mind the possibility of alkylation of coenzymes, especially the 

 nicotinamide nucleotides. 



Reactions with Proteins 



Protein SH groups are frequently resistant to iodoacetate and their reac- 

 tivity is usually increased by denaturation. Denatured and reduced oval- 

 bumin reacts with iodoacetate at pH 7.5 when the iodoacetate concentra- 

 tion is above 2.5 milf, and Rapkine (1933 b) showed that a 1 to 1 reaction 

 between iodoacetate and the SH groups occurs, so that this method can 

 be used to titrate the protein SH groups. The necessity of denaturation for 



