Our laboratory has characterized a family of peptides know as Peptide Inhibitor of Complement C1 (PIC1) that inhibit the classical pathway of complement by binding the initiator molecule C1q and inhibiting activation of the cognate serine protease tetramer C1s-C1r-C1r-C1s that is normally associated with C1q [7, 8].
PIC1 (IALILEPICCQERAA-dPEG24) was manufactured by PolyPeptide Group (San Diego, CA) to [greater than or equal to] 95% purity as verified by HPLC and mass spectrometry analysis.
Inhibition of peroxidase activity was assessed by combining the RBC lysate (reported in cellular equivalents/ml), metHb (0.5 mg/ml), or myoglobin (0.5 mg/ml) with increasing concentrations of PIC1, with a constant final volume, for 5 minutes before adding TMB as described below.
To assess if PIC1 could inhibit peroxidase activity of Hb released from hemolyzed RBCs in human serum, type O sera at 15% in GVBS++ buffer was mixed with type AB RBCs at titrating concentrations (1:2 dilutions of 5 x [10.sup.7] RBCs) followed by incubation for 1 hour at 37[degrees]C to allow for RBC hemolysis via antibody mediated complement lysis .
The oxidized TMB was transferred to a fresh well containing either water or 2 mM PIC1. The reaction was stopped after 1 min with 2.5 M [H.sub.2]S[O.sub.4].