Fig. 11.

Recombinant Amblyomma americanum serpin (rAAS) 41 inhibits and forms covalent complex with rat mast cell protease 1 (rMCP-1). (A) Inhibition of rat mast cell protease-1 (rMCP-1) activity from peritoneum-derived mast cells (PDMC) protein extract (1 U of chymase activity) was incubated with rAAS41 (1 μM) at 37 °C for 15 min following addition of substrate N-Succinyl-Ala-Ala-Pro-Phe-pNA (0.2 mM). Protease kinetics was monitored for 15 min with reads at every 11 s in triplicate. MW = molecular weight, MM = molecular marker, C+ = positive control. (B) Purified rAAS41 and rMCP-1 complex was resolved by 12% SDS-PAGE and visualized by silver staining. As controls, the gel was loaded with a protein extract containing rat chymase isolated from PDMC (1) and A. americanum serpin AAS41 (2). Complex formation (in red arrow) was evaluated by incubating equal volume of PDMC with AAS41 (4 μg) at 37 °C for various time points: 5 min (3), 15 min (4), 30 min (5), 45 min (6) and 60 min (7) and treated with deglycosylation enzyme mix. The enzyme deglycosylation mix only was loaded as a control (8). (C) Zymography was performed using 10% SDS-PAGE containing 0.1% casein. Gel was loaded with a protein extract containing rMCP-1 activity isolated from PDMC only (1) and rAAS41 only (2) as controls. Inhibition of rMCP-1 was performed by incubation with equal volume of PDMC with rAAS41 (4 μg) at 37 °C for different times: 5 min (3), 15 min (4), 30 min (5), 45 min (6) and 60 min (7).