FERRICYANIDE 671 



tential of the ferricyanide-ferrocyanide couple is + 0.36 v and does not 

 change from pH 4 to 10. The potential is, however, rather strongly depen- 

 dent on ionic strength. The potential is thus sufficiently high for SH groups 

 to be oxidized completely if they are available to the ferricyanide, and the 

 reaction is generally quite rapid. 



Most commercial preparations of ferricyanide contain ferrocyanide, which 

 may be detected by the Prussian blue method, and the latter should be re- 

 moved by addition of a little bromine water if SH determinations are done 

 by the colorimetric technique. Solutions of ferricyanide should be stored in 

 the cold and dark to avoid changes. 



Oxidation of Thiols and Proteins 



The reactions of ferricyanide with proteins have direct bearing on the 

 effects of ferricyanide on enzymes so that it is necessary to discuss the re- 

 sults in some detail. Although titration of cysteine and glutathione with 

 ferricyanide is rapid and provides good end-points, reactions with proteins 

 may not be so clear-cut. The conditions for the reaction are very important. 

 There is a marked effect of pH, as shown originally by Mirsky and Anson 

 (1936 a) for hemoglobin (see tabulation). Indeed, at pH 6.8 one may spe- 



pH Total SH groups oxidized (%) 



cifically oxidize the heme iron to form methemoglobin without affecting SH 

 groups. The conditions for reaction were: 83 mill ferricyanide incubated with 

 the protein for 30 min at room temperature — all reactive SH goups are 

 oxidized, as shown by titration of SH groups in denatured globin. Kolthoff 

 and Anastasi (1958) have also observed that oxidation of the SH groups of 

 denatured seralbumin is faster at pH 9 than 7. They noted that the reac- 

 tion is accelerated by Cu++, and Katyal and Gorin (1959) found in a study 

 of ovalbumin that iodide also catalyzes the oxidation by ferricyanide. 



Ferricyanide is not specific for SH groups, however, unless the conditions 

 are rigorously controlled, as shown early by Mirsky and Anson (1936 b) in 

 proteins not containing cysteine (zein and serum globulin) but nevertheless 

 reducing ferricyanide, or in proteins previously treated with cystine to oxi- 

 dize all the available SH groups. These groups are oxidized more slowly than 

 the SH groups and are more difficult to oxidize (e.g., milder oxidants than 



