Metal binding and ferritin immunoreactivity in a high molecular weight fraction from rat brain

A high molecular weight protein fraction (PI) from rat brain that exhibited heat- and acid-resistant properties like ferritin was characterized with respect to its reactivity with spleen ferritin antiserum, iron (Fe) content and Fe and aluminum (Al) binding. This fraction eluted on Sepharose 4B and Sepharose 6B columns with an apparent Mr of 2 100 000. A second Fe-rich fraction (PII) with an Mr of 500 000 showed lower anti-ferritin activity. The PI and PII fractions from liver, when prepared without the heat and acid steps, were similar to those found in brain. In both tissues, the PI fraction had a low Fe/ferritin ratio (0.28 +/- 0.08 microgram/microgram for brain and 0.22 +/- 0.05 microgram/microgram for liver). Similarly the PII fractions had high Fe/ferritin ratios (1.48 +/- 0.44 microgram/microgram for brain and 1.34 +/- 0.10 microgram/microgram for liver). Analysis of Fe binding to brain PI revealed one high affinity site (Kd = 157 +/- 30.0 X 10(-6) M; Bmax = 1.42 +/- 0.11 micromol Fe/mg protein) and one low affinity site (Kd = 890 +/- 100 X 10(-6) M; Bmax = 5.24 +/- 0.96 micromol Fe/mg protein). Removal of Fe from brain PI with 1% thioglycollic acid solution prior to Fe binding, increased binding affinity at both sites, while total binding remained unchanged. A1 bound to brain PI with high affinity (Kd = 59.7 +/- 13.6 X 10(-6) M; Bmax = 0.24 +/- 0.02 micromol A1/mg protein). These results demonstrate the presence of two ferritin-like, metal-binding (MB) proteins in rat brain.