Figure 5

Membrane vesicle aggregation by different AnxA2 derivatives. (A) Time-dependent changes in optical density at OD₆₀₀ (i) or OD₄₀₀ (ii) during aggregation of LUVs by non-modified WT AnxA2 (oxidation state after 10 weeks), alkylated WT AnxA2 and the N-terminal truncation mutants AnxA2 Δ14 or Δ32. LUVs without addition of protein served as control. 60 µl of the respective protein sample (0.5 mg/ml) were added at t = 300 s to a 800 µl LUV suspension at a concentration of 0.1 mg/ml (i) or 1 mg/ml (ii) in HBS buffer supplemented with 250 µM Ca²⁺. Monitoring continued for 55 min. In (i), 650 µM EGTA in HBS buffer was added at t = 3000 s to revert any Ca²⁺-dependent aggregation. In (ii), EGTA was included in a parallel recording to reveal a potential Ca²⁺-independent aggregation. (iii) shows a comparison of the sensitivity of the assay at 400 and 600 nm using the same amount of LUVs (0.1 mg/ml). (B) Size distribution of particles present using dynamic light scattering (DLS). 60 µl of the respective AnxA2 protein (0.5 mg/ml) were added to 800 µl of a LUV suspension (1 mg/ml; size of 150 to 200 nm) in HBS containing 250 µM Ca²⁺ or 650 µM EGTA (when indicated) under non-reducing conditions. The size distribution after 60 min compared to a vesicle alone control (green) was analyzed for non-modified WT AnxA2 (oxidation state after 10 weeks), alkylated WT AnxA2 and the N-terminal truncation mutant AnxA2 Δ32 (i). In (ii), vesicle aggregation by oxidized WT AnxA2 was compared for two LUV preparations, appr. 50 or 200 nm in size using the same non-reducing conditions. A comparison of DSC measurements under oxidizing and reducing conditions is shown in (iii) where TCEP (50 mM) was included as a reducing agent in one of the recordings. Data were evaluated using a CONTIN algorithm. Graphics shown are representative example of a typical measurement. Optical density measurements were performed two times with different batches of proteins and DLS measurements were carried out at least three time with two different batches of proteins and LUVs.