Metal ion binding to apo, holo, and reconstituted horse spleen ferritin.

This study examines how different metal ions interact with three forms of horse spleen ferritin. It was found that cadmium, zinc, and copper had the highest binding levels, while manganese, magnesium, and iron exhibited lower levels. The binding ability of most metal ions significantly decreased at a lower pH, with the notable exceptions of iron and copper. The amount of phosphate present also notably influenced the binding of the metal ions.

Methodology

• The researchers measured the binding of several metal ions (Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, Mg2+) to different forms of horse spleen ferritin (apo, holo, reconstituted) and native holo ferritin with phosphate removed. The binding was measured using gel-exclusion chromatography, a technique that separates molecules based on their size.

Main Findings

• For the various metal ions studied, three types of strong binding interactions were found with the apo Horse Spleen Ferritin (HoSF) at a pH of 7.5. The metal ions released different numbers of protons when bound, affecting pH levels.
• Cadmium, zinc, and copper had high binding levels, releasing two protons per metal bound.
• Nickel and cobalt showed intermediate binding, releasing one proton per metal ion bound.
• Meanwhile, manganese, magnesium, and iron demonstrated low levels of binding, releasing less than 0.5 protons per metal ion bound.
• The binding ability of these metal ions to the apo HoSF significantly decreased when the pH was lowered to 5.5, except for iron and copper, which remained unaffected by the pH alteration.

Influence of Phosphate

• Phosphate presence in HoSF had a considerable effect on metal ion binding. The holo form of HoSF showed a much higher level of metal ion binding ability, a result directly attributed to the presence of phosphate binding sites.
• This was further confirmed when less metal ion binding was witnessed in HoSF reconstituted without the presence of phosphate and when native holo HoSF had phosphate chemically removed.

Binding to Ferritin Interior

• The study found that cadmium alone among the tested metal ions was bound to the inside surface of the HoSF. The binding of cadmium to apo HoSF was 54 per ferritin molecule, but this number decreased when a ferritin ‘core’ was developing. This suggests that cadmium and the ferritin core compete for the same binding sites.
• No other tested metal ions appeared to bind to the interior of the HoSF according to this criterion.

Conclusions

• The researchers concluded that several of the tested metal ions appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF. This finding provides more insights into the role of both pH levels and phosphate presence in the binding ability of different metal ions to horse spleen ferritin.