Figure 4.

Monomer–dimer equilibrium of WT and variant ANX A13a in solution. A, evaluation of the elution profile of WT ANX A13a at 1 and 1.8 mg/ml. WT ANX A13a eluted as a doublet, with calculated molecular masses of 37.9 ± 3.7 and 74.5 ± 6.4 kDa. These values are consistent with the theoretical molecular masses of an ANX 13a monomer (35.4 kDa) and dimer (70.8 kDa). At a concentration of 1 mg/ml (blue trace), the monomer:dimer ratio is 4:1, whereas increasing the concentration to 1.8 mg/ml (orange trace) increases the percentage of the dimer such that the monomer:dimer ratio is now 1.5:1. Analysis was done in triplicate with a representative replicate shown. B, evaluation of the elution profile of 1 mg/ml wild-type ANX A13a at pH 7.0, 7.5, and 8.5. The peak corresponding to the ANX A13a dimer is reduced as pH increases. Analysis was performed in triplicate with a representative chromatogram shown. C, comparison of dimerization propensity of purified WT and variant ANX A13a at a concentration of 0.5 mg/ml. WT ANX A13a (blue trace) exhibits a distinct monomer:dimer distribution. The ANX A13a^{T32P/N33G/E34P} (green trace) designed monomer exhibits no detectable dimer. The ANX A13a^S73C (orange trace) designed dimer exhibits an increase in dimerization over WT but is not fully converted to the dimeric form under the conditions tested. Analysis was performed in triplicate with each replicate shown.