Figure 5.

Chromatogram for the separation of nonentrapped HRP from HRP-encapsulating POPC LUV₂₀₀ by size exclusion chromatography with Sepharose 4B (l = 35 cm; d = 1.1 cm; applied volume, 0.5 mL (≈12 mM POPC, 31 μM HRP) (polycarbonate membrane extrusion resulted in a loss of POPC of ≈38%. Therefore, the POPC vesicle suspension applied onto the column had a concentration of ≈12 mM instead of 20 mM.); elution flow rate (buffer-2), 0.33 mL/min; fraction volume, 0.78 mL), see the Experimental Section for details. (a) A₂₇₅ and A₄₀₃ vs fraction number (l = 1 cm); (b and c) HRP activity measured with ABTS²⁻ (b) or PPD (c) without (blue, open squares) and with cholate (8.0 mM, black, filled circles); the y-axis on the left-hand side is the increase in A₄₁₄ (for ABTS²⁻) or A₅₀₀ (for PPD) per s, caused by the addition of 10 μL of the fraction to 1 mL of the assay solutions (l = 1 cm); the axis on the right-hand side refers to the calculated amount of HRP for the measurements carried out in the presence of 8.0 mM cholate and taking into account the calibration curves in Figure S-5b (for ABTS²⁻) and Figure S-8b (for PPD); (d) POPC concentration, as determined with the Stewart assay and taking into account the calibration curve in Figure S-3 (left y-axis, red); the axis on the right-hand side refers to the calculated number of vesicles, see Table 1.