Activation and Assay of Prophenoloxidase Cascade

To activate procSP_Xa with factor Xa, purified procSP_Xa was incubated with bovine factor Xa (New England Biolabs, Ipswich, MA, United States) in buffer [20 mM Tris–HCl, 0.1 M NaCl, 2 mM CaCl (pH 8.0)] at 27°C for 5 h. Amidase activity of the reaction mixtures was measured using 200 μL, 50 μM acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (IEAR) as the substrate (39 (link)). One unit of amidase activity was defined as ΔA405 of 0.001 in one minute. Factor Xa activated procSP_Xa was incubated with procSP at 37°C for 1 h before immunoblot analysis under reducing conditions containing β-mercaptoethanol (β-ME) or non-reducing conditions. Mixtures containing sequentially activated SP cascade components (cSP6_Xa, cSP1_Xa/procSP6, and SP41_Xa/procSP1/procSP6) were incubated with purified PPO at room temperature for 10 min to detect PPO cleavage by immunoblotting. To measure PO activity, samples were transferred to 96-well plates, and 200 μL of 2 mM Dopa in 50 mM sodium phosphate buffer (pH 6.5) were added. The activity was determined by measuring the absorbance at 470 nm with a microplate reader (Synergy H1; BioTek, Winooski, VT, United States). One unit of PO activity was defined as ΔA470 of 0.001 in one minute (30 (link)).