Bovine factor xa

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)).

To test whether proSP105_Xa could be activate by Factor Xa, 0.25 μg of purified recombinant proSP105_Xa was incubated with 0.2 μg of bovine Factor Xa (New England Biolabs) in the reaction buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM CaCl₂, pH 8.0) at 37 °C for 4 h. The mixtures were separated by 10% SDS-PAGE followed by immunoblot analysis with anti-His as primary antibodies.
The activation of proSP105_Xa was confirmed by measuring the amidase activity of activated SP105_Xa with 200 μl of 50 μM acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (IEARpNA) in 0.1 M Tris-HCl (pH 8.0), 0.1 M NaCl and 5 mM CaCl₂ as colorimetric substrate. The amidase activity was measured by monitoring changes in absorbance at 405 nm in a microplate reader (Bio-Tek Instrument, Inc.). One unit of amidase activity was defined as ΔA₄₀₅/min = 0.001.

To activate recombinant CLIPB9_Xa and CLIPB10_Xa, 2.5 µg of each purified zymogen was incubated with 1 µg of bovine Factor Xa (New England Biolabs) in a total volume of 50 µl in reaction buffer (20 mM Tris, 100 mM NaCl, 2 mM CaCl₂, pH 8.0) at 37°C overnight. Two negative controls were set up in parallel, in which either Factor Xa or the zymogen was replaced with same volume of buffer. Cleavage of the zymogen was examined by loading 8 µl of the activation reaction to 10% SDS-PAGE followed by Coomassie blue staining for visualization.