672 5. OXIDANTS 



ferricyanide will not oxidize them), but their total reducing capacity (the 

 amount of ferricyanide they can reduce) is often greater than for the SH 

 groups. Furthermore, the rate and degree of oxidation of these non-SH 

 groups depend on the same factors as reaction with SH groups; thus, the 

 rate is accelerated by rise in pH, rise in temperature, and denaturation, 

 Native ovalbumin is not oxidized at all bj^ ferricyanide, but denatured oval- 

 bumin treated with cystine to remove SH groups reduces ferricyanide. In 

 other words, these other groups become available during unfolding of the 

 protein. /5-Lactoglobulin, which contains 2 SH grovips per molecule (molec- 

 ular weight of 37,000), reacts very slowly with ferricyanide in the native 

 state but rapidly in the presence of urea or guanidine (Leslie et al., 1962 a). 

 The stoichiometry indicates that the SH groups are oxidized beyond the 

 disulfide stage. Since the reaction was carried out under fairly mild condi- 

 tions (0.1-0.8 mM ferricyanide, pH 7, 37^, and 30-45-min incubation), it 

 is evident that one cannot generally assume the simple formation of disul- 

 fides from the actions of ferricyanide on enzymes. Ferricyanide can oxidize 

 tyrosine and tryptophan, but not histidine, and the characteristics of the 

 oxidation parallel oxidations of proteins. Mirsky and Anson suggested that 

 tyrosine and tryptophan are the residues responsible for ferricyanide re- 

 duction. Gelatin, which contains no (or very little) tyrosine and no tryp- 

 tophan, scarcely reacts with ferricyanide, supporting this view. Anson 

 (1939 b) observed that at pH 9.6, where much previous work had been 

 done, the oxidation is nonspecific, but that at pH 6.8 combined with the 

 treatment of the protein with Duponol PC the SH groups react rapidly and 

 specifically if not too much ferricyanide is used (2-5 mM is best); under 

 these conditions there is no reaction with cystine, tyrosine, tryptophan, or 

 proteins that do not contain cysteine. The specificity of SH group oxida- 

 tion could also be shown by pretreatment of denatured ovalbumin with 

 iodoacetamide, following which ferricyanide is no longer reduced by the 

 protein. Katyal and Gorin (1959) also demonstrated such specificity by 

 blocking SH groups with p-chloromercuribenzoate. Mirsky (1941) discussed 

 the method in detail and showed that when properly run the oxidation oc- 

 curs within 1 min. Barron (1951) has also reported in detail his modifica- 

 tion of the method. Various oxidations by ferricyanide have been reviewed 

 by Thyagarajan (1958). 



Inhibition of Enzymes 



One must conclude from the results with proteins that application of ferri- 

 cyanide to enzymes cannot be done haphazardly if specific effects on SH 

 groups are to be anticipated. Unfortunately most studies have used ferri- 

 cyanide along with numerous other inhibitors under the same conditions 

 of pH, temperature, and incubation time, without considering that rather 

 stringent conditions have been proposed for the use of ferricyanide. Some 



