ABSTRACT
The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. Ferritin iron loading in imidazole shows a rapid hyperbolic curve in contrast to iron loading in 3-(N-morpholino)propanesulfonic acid (MOPS), which displays a slower sigmoidal curve. Ferritin iron loading in an equimolar mixture of imidazole and MOPS produces an iron-loading curve that is intermediate between the imidazole and MOPS curves indicating that one buffer does not dominate the reaction mechanism. The UV-visible spectrum of the ferritin mineral has a higher absorbance from 250 to 450 nm when prepared in imidazole buffer than in MOPS buffer. These results suggest that different mineral phases form in ferritin by different loading mechanisms in imidazole and MOPS buffered reactions. Samples of 1500 Fe/ferritin were prepared in MOPS or imidazole buffer and were analyzed for crystallinity and using the electron diffraction capabilities of the electron microscope. The sample prepared in imidazole was significantly more crystalline than the sample prepared in MOPS. X-ray powder diffraction studies showed that small cores (~500 Fe/ferritin) prepared in MOPS or imidazole possess a 2-line ferrihydrite spectrum. As the core size increases the mineral phase begins to change from 2-line to 6-line ferrihydrite with the imidazole sample favoring the 6-line ferrihydrite phase. Taken together, these results suggest that the iron deposition mechanism in ferritin can be controlled by properties of the buffer with samples prepared in imidazole forming a larger, more ordered crystalline mineral than samples prepared in MOPS.
