Figure 3.

Tetrapyrrole inhibition of HCV NS3/4A protease. (A,B) Protease activity was determined fluorometrically (FRET assay) using recombinant NS3/4A enzyme and various concentrations of inhibitors. (C) Endogenous NS3/4A protease activity in microsomes of replicons was measured using the same FRET assay but employing endogenous, partially purified NS3/4A protease from replicon cells (Zhu et al., 2010a). (D) Reciprocal (Lineweaver–Burk) plot of substrate concentration vs. enzyme activity. Recombinant protease activity was determined fluorometrically. Each point is the mean ± SEM of 3–5 determinations per point. Plot of [BV] vs. either 1/Vap or Km/V (not pictured) showed highly significant linearity (r = 0.975 and r = 0.979 respectively, p < 0.005) indicating mixed inhibition of NS3/4A protease by BV (${K^{\prime }}_{\text{i}}$ = 1.1  mM and K_i = 0.6 mM, respectively). 0 to the commercial Inhibitor = NS3/4A protease competitive inhibitor, AnaSpec #25346. Biliverdin = >99% Biliverdin IX-α. Bilirubin-mixed isomers (MI) = 93% Bilirubin IX-α, and 6% associated Bilirubin isomers. Bilirubin IX-α = >99% bilirubin IX-α. (With permission Zhu et al., 2010a see original manuscript for further details.)