Stage-specific expression of FP-2.
(A) Giemsa-stained smears of highly synchronized parasites at ring (i), early trophozoite (ii), late trophozoite (iii), and schizont (iv) stages. (B) Immunoblot analysis of rFP-2 (lane 1) and soluble parasite extracts from 1 × 108 parasites/lane (lanes: i, rings; ii, early trophozoites; iii, late trophozoites; iv, schizonts) using antibodies raised against rFP-2. (C) Gelatin substrate analysis: rFP-2 (lane 1) and soluble parasite extracts from 1 × 108 parasites/lane were analyzed by 10% gelatin substrate SDS-PAGE. Protease activity at pH 5.5 and at pH 7.0 was identified as clear bands against a Coomassie blue–stained background. (D) Cleavage of human erythrocyte membrane skeletal proteins (left) and hemoglobin (right): 5 μL human erythrocyte IOVs (2 mg/mL) (lane 1′, left panel) in 5 mM sodium phosphate buffer, pH 7.0, 1 mM DTT or 3 μg human hemoglobin (lane 1′, right panel) in 100 mM sodium acetate, pH 5.5, and 1 mM DTT were incubated with 0.2 μM rFP-2 (lanes 1) or identical volumes of soluble parasite extracts for 30 minutes (IOVs) or 60 minutes (Hb) at 37°C, followed by SDS-PAGE analysis.
