Internal Structure of Apoferritin as Revealed by the "Negative Staining Technique."

I t has been surmised that the apoferritin molecule has a hollow center which communicates with the outer surface through some sort of channels or slots (1-2). This inference is based upon the following findings: 1. As determined by means of electron microscopy, the ferric hydroxide (FeOOH) micelles of ferritin molecules are located within spheroids of apoferritin (1) ; 2. In reducing media, such as dithionite solutions, iron can be removed with ease from ferritin, intact apoferritin being left behind (4); 3. Apoferritin, prepared following removal of iron from ferritin, has the immunologic specificity of ferritin, crystallizes in the same habit as ferritin, and has the molecular crystal lattice parameters of ferritin (3-8). If the ferric hydroxide micelles in the ferritin molecule were tightly enclosed by a shell of the protein, apoferritin, their removal would require some kind of unfolding of the protein---/.e, denaturation. Such denaturation would be inconsistent with the facts just summarized. On the other hand, if there are communications between the outside and the inside of the apoferritin shell, then various solvents and solutions should penetrate readily into the interior of ferritin molecules. I t follows that with suitable solutions the reduction of the ferric hydroxide micelles to ferrous ions could be brought about in the interior of ferritin molecules; ferrous ions would then diffuse outward. This hypothesis is consistent with the facts summarized above, and with the findings reported by Bielig and Bayer (9) and by Loewus and Fineberg (10); viz. that ferritin can be reconstituted from apoferritin and ionic iron in vitro. Like the molecules of many proteins, those of apoferritin are not sufficiently dense to electrons to be visible directly in the electron microscope. However, with the negative staining technique of Brenner and Home (11), we have been able to demonstrate the contours of apoferritin molecules. In this technique phosphotungstic acid is used to