Formation of SDS-stable complexes between thrombin and N-terminal or C-terminal HV3 54-66–API M358R fusion proteins. Polypeptides comprising portions of API M358R (hatched bar, with residue numbers above) with or without C-terminal extensions (white) derived from HV3 (with single “minus sign” symbols inset, and residue numbers below) are represented schematically in Panel A. Each protein contains an N-terminal MGSH6 tag (shown at left of each schematic representation as a thin grey bar). Fusion proteins contain (API-G6-HV3) or do not contain (API-HV3) a six residue spacer peptide (thin black bar) between C-terminal extension and API M358R body identified above the bar. Reactive centre loop (RCL) sequences are API M358R (open box) in both cases. Panel B shows a reduced 10% SDS polyacrylamide gel stained with Coomassie Brilliant Blue. Proteins identified above the lanes were incubated (1.0 μM serpin, 0.2 μM thrombin) without (-) or with (+) thrombin (IIa) at ambient temperature for 1.0 minute. The position of the serpin-enzyme complex (SEC) is highlighted with arrowheads. The position of unreacted serpin (S), cleaved serpin (CS) and the B chain of thrombin (IIa (B)) is shown at right. M indicates molecular mass markers of 160, 120, 100, 90, 80, 70, 60, 50, 40, and 30 kDa, respectively. Panel C shows a plot of the second order rate constants of thrombin inhibition (k2, in units of 106 M-1 min-1) of the purified proteins identified, in each case, below the x axis. The mean ± SD for 9, 6, and 5 replicated determinations is shown. Lines between the columns indicate statistical significance (non-parametric ANOVA, Kruskal-Wallis test with Dunn’s post-tests): *, p < 0.05; ***, p < 0.001; n.s., not significant).