Figure 4

Native PAGE and overlay of size-exclusion chromatograms of S100A12 in the presence of EDTA and different cations. Loading concentration of S100A12 – 14 μg (A-E). Dimeric S100A12 is indicated by an arrow. Concentrations of zinc acetate and calcium chloride are indicated above the gel picture. (F) TPEN (zinc-chelator), BCS (copper I-chelator), or EDTA (divalent cation chelator) was added. After removal of calcium or zinc from the buffer solution the dimeric S100A12 is the strongly preferred form, while removal of copper did not produce a measurable effect. (G) S100A12 in a zinc- and calcium-free solution was re-substituted with zinc, copper, or calcium before loading the gel. There were no hexamers in the absence of zinc (w/o), which is also not influenced by addition of copper. When re-substituting zinc, there is predominantly a hexamer formation, which is also induced to a lesser extent by adding calcium, where a second band eventually representing tetrameric S100A12 can be observed. (H) SEC was performed using Akta FPLC chromatography equipment (Amersham Pharmacia Biotech) and a Superdex 200 (10 mm×300 mm) gel-filtration column (Amersham Pharmacia Biotech). Before each run the column was equilibrated with elution buffer 50 mM Tris, pH 7.5, 200 mM NaCl. The flow rate was 0.5 ml/minute and the protein elution was monitored by UV-absorption at 280 nm. S100A12 Abs 280/260 = 0.4/0.3. S100A12 elutes as a dimer in the presence of EDTA (shown in violet), shows a tendency to oligomer formation at increasing calcium concentrations (red and blue curves), elutes as a tetramer in the presence of zinc (orange), and as a hexamer with calcium and zinc (green).