Figure 3. Characteristics of Re-LPS aggregation induced by SP-A.

(A) Extent of SP-A-induced Re-LPS aggregation as a function of Re-LPS concentration. Different kinetic assays were performed in which increasing amounts of Re-LPS were added to both the sample and reference cuvette in 5 mM Tris/HCl buffer (pH 7.4), containing 150 mM NaCl and 0.2 mM EDTA at 37 °C. After 10 min equilibration at 37 °C, 10 μg/ml SP-A was added to the sample cuvette in each experiment. Re-LPS aggregation induced by SP-A started after the addition of 2.5 mM calcium to both the sample and reference cuvette. The contribution of self-association of SP-A (10 μg/ml) to the turbidity change at 400 nm was negligible. (B) Extent of SP-A-induced Re-LPS aggregation as a function of SP-A concentration. Different kinetic assays were performed as described in (A), but increasing amounts of SP-A were added to both the sample and reference cuvettes to eliminate the contribution of self-association of SP-A to the light absorption change at 400 nm. Re-LPS aggregation induced by SP-A started after the addition of 4×10⁻⁶ M Re-LPS (20 μg/ml) to the sample cuvette and Ca²⁺ (2.5 mM) to both the sample and reference cuvettes. (C) Ca²⁺ dependence of SP-A-induced Re-LPS aggregation. Titration experiments were performed at 37 °C as described in the Experimental section. (A–C) Results of one representative experiment from three experiments performed are shown.