The effect of the putative nucleation sites on the loading and stability of iron in ferritin

Ferritin L chain contains more putative nucleation sites (Glu53. 56, 57, 60, and 63) than ferritin H chain (G!u6l, 64. and 67) This study was to investigate the role of the putative nuclealion sites on iron loading and the stability of the iron core of turrit in Site-directed mutagenesis techniques were used to obtain femtin II chain homupolymer containing 2 or 1 proposed nucleation sites Recombinant ferritin II chain homopolymer and the two mutants (I-61A and E61A and E64A) loaded iron up to 1950 ±20, 2010 i 40, and 2010 ±10 atoms of iron per territin, respectively. However, the mutations resulted in a 50% decrease in the rate of iron loading by ceruloplasmin These fenitins also incorporated the same amount of phosphate after iron loading (420 t 20, 400 t 30, and 410 ±20 atoms per ferritin, respectively) The stability of the iron coies, assessed by the rate of iron release by 1 mM Desferal and the paraquat radical, corresponded to the numbers of proposed nucleation sites present in ferrit in II chain honiopolymei The subsequent incorporation of phosphate in ferritin following loading by ceruloplasmin icsulted in stabilizing the iron core even further. Therefore, the numbeis ut"proposed nucleation sites may not affect the amount of iron or phosphate loaded, however, they indeed affected the stability of ferritin iron core (Supported by NIH Grant ES05056.).