The specific activity of IRPs, both before and after purification through an IRE-affinity column, can be determined through the use of electrophoretic band-shift assays. (A) A constant concentration of 50 nM of 32P-labeled C-IRE was treated with increasing concentrations of nonaffinity-purified IRP1, then electrophoresed on a 10% nondenaturing polyacrylamide gel. Autoradiography revealed that the specific binding activity of IRP1 was approximately 30%. Although not shown, nonaffinity-purified IRP2 had even lower specific activity. (B) Affinity-purified IRP1 was analyzed in an experiment similar to that shown in A. Autoradiography revealed at least a twofold improvement in the specific activity in comparison to nonaffinity-purified protein. (C) Affinity-purified IRP2 was analyzed in a similar fashion to reveal an apparent specific activity of ∼50%, which was substantially greater than the activity of nonaffinity-purified protein (not shown).
