16 1. lODOACETATE AND lODOACETAMIDE 



ovalbumin does not react discernibly in 5 hr, indicating that both SH and 

 amino groups must be protected, but Anson (1940) reported that iodoacet- 

 amide at 19 mM and pH 9 would carboxymethylate 40% of the SH groups 

 in 30 min, even though these groups do not react with porphyrindin or 

 ferricyanide. Anson considered the SH groups in the native protein to be 

 accessible but of low reactivity. A spectrophotometric method was used by 

 Finkle and Smith (1958), based on differences in the absorbance of iodo- 

 acetate and iodide at 235 mju and 275 m//, and Watts et al. (1961) developed 

 an iodide electrode for measuring the iodide formed in the reactions of iodo- 

 acetate with proteins. The latter found, incidentally, that urea-denatured 

 ovalbumin reacts rapidly with 2 equivalents of iodoacetate, and then slowly 

 until a further 9.8 equivalents are used. 



It has generally been found that protein amino groups react very slowly 

 or not at all with iodoacetate under the conditions of most enzyme work. 

 Hirade and Ninomiya (1950) attempted to correlate the toxicity of iodo- 

 acetate and other alkylating agents with the protein groups reacted, and 

 concluded that SH groups alone were of importance, inasmuch as amino 

 groups reacted very slightly under conditions (pH 8.4 and 30°) where glu- 

 tathione reacted rapidly and completely. Likewise Habeeb (1960) found /?- 

 lactoglobuhn to react with iodoacetate below pH 8.5 in a rapid and a slow 

 phase, the latter lasting up to 30 hr and possibly due to amino groups. Of 

 course, under more rigorous conditions it is possible to alkylate amino 

 groups, as shown by Korman and Clarke (1956 a, b), who introduced the 

 carboxymethyl group into amino acids and proteins by using bromoacetate 

 at 200 mM at pH 9 and 35° and incubating for 2-3 days. Such carboxy- 

 methylation involves histidine and tyrosine residues and is akin to denatura- 

 tion since disulfide groups are made available to sulfite. A similar technique 

 was used by Nakatani (1960 a, b) to carboxymethylate hemoglobin. The 

 optimal pH for reaction of histidine groups is pH 9 and tyrosine reacts very 

 slowly under these conditions. Reaction stops within 10 hr at 30^ and about 

 60% of the histidine residues are modified; treatment with urea leads to 

 some further reaction. About one third of the 95 histidine residues in catalase 

 react with bromoacetate by this procedure. 



Milder alkylating conditions (incubation with 36 mM iodoacetamide for 

 1 hr at pH 7.15 and 25°) were used by Guidotti and Konigsberg (1964) to 

 study the role of the SH groups in human hemoglobin. It appears that the 

 amino groups do not react readily since only 5-carboxymethylcysteine was 

 found in hydrolyzates. The SH groups which influence heme-heme interac- 

 tion are located next to the histidine residues linked to the iron atoms of 

 the hemes, but 4 of the 6 SH groups in the native carbonmonoxyhemoglobin 

 do not react with iodoacetamide. It has been thought that these unreactive 

 groups are inside the tetrameric structure, but it was shown that the mon- 

 omers likewise are not attacked, so it was suggested that the SH groups 



