Figure 2.

Ca²⁺ stabilizes both oxidized and reduced LbPrx1m decamers. aSEC chromatograms of LbPrx1m at different concentrations in Tris buffer (pH 7.5) containing 5 mm EDTA (a), 5 mm EDTA with 10 mm DTT (b), 25 mm CaCl₂ (c), or 25 mm CaCl₂ with 10 mm DTT (d). Roman numerals represent the protein concentration of the input samples (250 μl): I, 94 μm; II, 23 μm; III, 9 μm. Numbers above the graphs represent the molecular mass (kDa) of standard proteins used for column calibration. For this assay, the His tag was removed, using TEV protease to show that the untagged protein behaves similar to the His-tagged samples upon CaCl₂ and EDTA treatments (see Fig. 1a). Note that the Ca²⁺-stabilized decamers, preformed at 94 μm protein, did not dissociate upon protein dilution even in the absence of DTT. Samples not treated with DTT are air-oxidized (S–S-bonded). mAU, milliabsorbance units.