Complex formation of radiolabeled granzyme A (GrA) to proteinase inhibitors in plasma.

Approximately 1 pg of ¹²⁵I-GrA was incubated with 2 μL of plasma for 30 minutes at 20°C. Next, samples were either prepared for SDS-PAGE directly or incubated with 2 mL monoclonal antibody (mAb) coupled to Sepharose beads (2 mg/mL) for 30 minutes followed by recovery of the Sepharose-bound proteins that then were prepared for SDS-PAGE. After electrophoresis, the gel was dried and labeled; GrA was visualized by autoradiography. (A) Complex formation of¹²⁵I-radiolabeled GrA in EDTA plasma: lane 1, GrA; lane 2, GrA incubated with EDTA plasma; lane 3, GrA incubated with plasma in the presence of phenylmethylsulfonyl fluoride; lane 4, immunoprecipitation of purified GrA with mAb M1; lane 5, immunoprecipitation of GrA in EDTA plasma with mAb M1; lane 6 and 7, as lanes 4 and 5 respectively, but under reducing conditions. (B) Complex formation of ¹²⁵I-radiolabeled GrA in heparin plasma: lane 1, GrA; lane 2, GrA incubated with heparin plasma; lane 3, GrA incubated in heparin plasma in the presence of phenylmethylsulfonyl fluoride; lane 4, immunoprecipitation of purified GrA in heparin plasma with mAb a-antithrombin III; lane 5, 6, 7, and 8 same as lane 1, 2, 3, and 4, respectively, but under reducing conditions.