204 Essays in Biochemistry 



and the antidiuretic action in hydrated dogs as indices of physiological 

 activity. 11 Inactivation of these ferritin activities by aerobic liver 

 slices (rat, rabbit, or dog) and activation by anaerobic liver slices sug- 

 gested the presence of groups in ferritin capable of undergoing revers- 

 ible oxidation-reduction. Inactive ferritin could be activated by treat- 

 ment with cysteine or reduced glutathione, whereas active ferritin was 

 inactivated by treatment with iodoacetamide, o-iodosobenzoate or p- 

 chloromercuribenzoate, all sulfhydryl-reacting reagents. These data 

 appeared to fit the hypothesis that sulfhydryl groups were involved 

 in these biological activities of ferritin. 



Chemical estimation of sulfhydryl groups yielded elevated values 

 for active ferritin and decreased values for inactive ferritin. The 

 choice of a method for measurement of SH content was a difficult 

 problem because of the intense color of the protein. Amperometric 

 titration with Ag+ or Hg++ gave poor results with ferritin as it does 

 with some proteins. The method of Rosner using iodoacetic acid was 

 modified so as to use iodoacetamide followed by precipitation of the 

 ferritin with trichloracetic acid. The extent of reaction of sulfhydryl 

 groups with iodoacetamide (for a 10-minute period at pH 7.4) was 

 measured in the clear supernatant solution by oxidizing the HI formed, 

 with H2O2, to yield free iodine. The iodine color was read in a photo- 

 colorimeter before and after treatment with thiosulfate. If adequate 

 blanks are used, this method gives reproducible values and is quite 

 specific for sulfhydryl groups since addition of p-chloromercuriben- 

 zoate prior to reaction with iodoacetamide reduces the extent of reac- 

 tion of ferritin with iodoacetamide, under these conditions, to zero. 



The conclusion, based on the data described above, that sulfhydryl 

 groups were directly related to ferritin activity, was brought into 

 question by an experimental finding contradictory to this hypothesis. 

 When inactive iodoacetamide-treated ferritin is incubated with cys- 

 teine, reduced glutathione or ascorbic acid, the resulting ferritin be- 

 comes quite active. Since the reaction of sulfhydryl groups with 

 iodoacetamide is known to be irreversible, we had to consider the pos- 

 sibility that another group capable of undergoing oxidation-reduction 

 might be responsible for activity of this protein. 



Iron and Ferritin Activity 



In our earlier studies, iron was not seriously considered as related 

 to ferritin activity for two reasons: 



(a) Apoferritin, essentially free of iron, is as active as ferritin con- 

 taining 23% iron, when tested in the rat for vasodepressor activity 



