Validation of site-directed PEGylation method. (A) Cartoon representation of full-length pseutarin C (A1, purple; A2, green; A3, beige; C domains, gray; and fXa, cyan), indicating the position of Cys540 (blue balls) and Ser578 (red balls) that were selected as controls to validate the PEGylation method. ptfV has a free cysteine residue (Cys540) at the back of the molecule (A2 domain, green). This residue was selected as a negative control, because PEGylation was not expected to affect prothrombin binding. Ser578 is located in the binding interface between ptfV and ptfXa (cyan) in the pseutarin C structure and was therefore selected as a positive control to test the effect of PEGylation on pseutarin C activity. Both positions were labeled with maleimide-PEG11-biotin as described in the Methods section. The effects of both labeled (blue squares) and unlabeled (red circles) Cys540 (B) and S578C (C) were tested for prothrombin processing using chromogenic (top) and SDS-PAGE (bottom) assays. As expected, PEGylation of Cys540 had no effect on thrombin generation, whereas PEGylation of S578C completely prevented processing of prothrombin by pseutarin C. II, prothrombin; F1.2-L, F1.2+light chain; IIa-H, thrombin heavy chain; mAu, milli–absorbance unit.