MOLECULAR-DIFFUSION INTO HORSE SPLEEN FERRITIN - A NITROXIDE RADICAL SPIN-PROBE STUDY

Electron paramagnetic resonance spectroscopy and gel permeation chroma tography were employed to study the molecular diffusion of a number of small nitroxide spin probes (similar to 7-9 Angstrom diameter) into t he central cavity of the iron-storage protein ferritin. Charge and pol arity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidin e- (4-carboxy-TEMPO) does not penetrate the cavity whereas the positiv ely charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radic al does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate tha t diffusion is not purely passive, the driving force coming not only f rom the concentration gradient between the inside and outside of the p rotein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21 -26 min are observed, suggesting that reductant molecules with diamete rs considerably larger than similar to 9 Angstrom would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.