NONHEMATIN IRON IN TISSUES 567 



the hematin colorimetrically ; it is doubtful whether this method gives 

 reHable values. 



10.3.5. Chemical Nature of Nonhematin Iron. Ferritin. The chemical 

 nature of nonhematin iron is not yet fully understood. The only well investi- 

 gated compound which forms a large part of the storage iron in the spleen, 

 the intestinal mucosa, and probably also in the liver, is ferritin, a crystallizable 

 protein containing 20-24% iron. Ferritin was first isolated from horse spleen 

 by Laufberger (1660) as a crystalline protein, by the use of cadmium sulfate. 

 It was later studied by Kuhn and co-workers (1619) and particularly by 

 Granick and Michaelis {103^1030,1037 J038, 1937). It is precipitated by 

 trichloroacetic acid and in this way little of its iron is removed; the iron is 

 removed, however, by pyrophosphate in trichloroacetic acid, or by reduction 

 of the ferric to ferrous iron with dithionite. According to Scott [2519) 23% 

 of ferritin iron is removed by thiocyanate in dilute hydrochloric acid. 



Ferritin contains 1.2-2% phosphorus which Kuhn assumed to be present 

 as nucleic acid; Granick, however, found no evidence for this. Ferritin is 

 stable at />H -1-10, but its iron is removed at pH 4.G by dithionite and 

 dipyridyl. By removal of the iron the colorless apoferritin is obtained which 

 crystallizes with cadmium sulfate in the same form and shape as ferritin. 

 The x-ray powder diagrams of ferritin and apoferritin crystals show the 

 same structure and identical cell size, the packing of the protein molecules 

 not being disturbed by the iron {737). This, together with the fact that the 

 iron can also be removed by ultracentrifugation. indicates that ferritin con- 

 tains micelles of colloidal ferric hydroxide in the interstices of apoferritin. 

 The conception of Behrens and Asher {202) that the iron is present in the 

 spleen as ferric hydroxide embedded in protein has thus reappeared in 

 modern dress. 



Michaelis and co-workers {1937) have shown that ferritin contains three 

 unpaired electrons per iron atom. In colloidal ferric hydroxide different 

 forms having from one to five free electrons per iron atom were found to 

 exist. This is interpreted as being due to partial dehydration which estab- 

 lishes oxygen bridges between iron atoms and causes neighboring octahedra 

 to share corners and edges. According to the valency angle on the oxygen 

 atom, the linkages may be covalent or ionic, the former if, for example, one 

 edge with two corners is shared. Such micelles of partly dehydrated ferric 

 hydroxide must be present in ferritin. Holden {1317) has criticized the mag- 

 netochemical evidence on the ground that the iron content of ferritin varies. 

 This criticism is unjustified, since the estimation of the paramagnetism was 

 based on the iron content. 



While apoferritin is entirely homogenous, ferritin is dishomogeneous in 

 the ultracentrifuge and in solubility experiments, but homogeneous in elec- 

 trophoresis. Ferritin can be regenerated from apoferritin. 



In rat liver and spleen Scott found a much smaller percentage of the storage 

 iron removable by means of thiocyanate in dilute hydrochloric acid than was 

 the case with ferritin, and concluded that the storage iron in the rat is not 

 ferritin. To judge from McFarlane's results {1813), it is more likely that 

 rats' liver contains a mixture of ferritin with another ferric ion compound; 



