The interaction of the recombinant catalytic domain of congopain (C2) with bovine α₂M. (A) The effect of increasing amounts of α₂M on the activity of C2 against hide powder azure (▲) and Bz-Pro-Phe-Arg-pNA (□). Different concentrations of α₂M were incubated with C2 (100 pmol for hide powder azure assay; 20 pmol for Bz-Pro-Phe-Arg-pNA assay) at molar ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 for 20 min at 37 °C. Proteolytic activity was determined by the extent of hydrolysis of substrate compared to that of a C2 control with no α₂M (100% activity), and expressed as a percentage inhibition. Error bars represent the ± SEM (n = 3). (B) Non-denaturing PAGE (5% gel) analysis of the interaction between bovine α₂M and activated C2. Lanes 1 and 9 500 ng α₂M; lanes 2–8, α₂M (500 ng) was incubated with increasing amounts of C2 (11–91 ng), corresponding to molar ratios of α₂M:C2 of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 in the respective lanes. Proteins were silver stained. (C) Reducing SDS-PAGE (7.5% gel) after reaction of bovine α₂M with activated C2. Lane 1, Bio-Rad molecular weight markers consisting of myosin (200 kDa), β-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa); lane 2, α₂M (10 μg); lane 3, C2-α₂M complex (10.372 μg). Proteins were stained with Coomassie blue R-250. Arrows represent bands at 95 and 85 kDa.