Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Ferritin is an iron-storage protein with a central cavity.
• Understanding molecular transport into ferritin is crucial for its biological function and potential applications.
• Nitroxide spin probes are useful tools for studying diffusion in biological systems.

Purpose of the Study:

• To investigate the molecular diffusion of small nitroxide spin probes into the ferritin protein cavity.
• To determine the role of charge and polarity in the diffusion process.
• To develop a model for ferritin diffusion kinetics.

Main Methods:

• Electron paramagnetic resonance (EPR) spectroscopy was used to detect and quantify spin probes.
• Gel permeation chromatography (GPC) was employed to assess probe size and diffusion.
• Various nitroxide spin probes with different charges and polarities were utilized.

Main Results:

• Negatively charged 4-carboxy-TEMPO did not penetrate the ferritin cavity.
• Positively charged (4-amino-TEMPO, 3-(aminomethyl)-proxyl) and polar (4-hydroxy-TEMPO) probes successfully diffused into the cavity.
• Apolar TEMPO bound to the protein surface instead of entering the cavity.
• Diffusion kinetics indicated a driving force beyond simple concentration gradients, involving charge interactions.

Conclusions:

• The charge and polarity of small molecules significantly influence their ability to diffuse into the ferritin protein cavity.
• Diffusion into ferritin is not purely passive, with electrostatic interactions playing a key role.
• Molecules larger than approximately 9 A are likely to diffuse too slowly for efficient iron mobilization within the ferritin core.