The kallikrein substrate H-D-Pro-Phe-Arg-p-nitroanilide was used in a direct spectrophotometric assay for activated Hageman factor (HF). An 80,000-dalton two-chain, disulfide-linked enzyme, termed HFa, and a 28,000-dalton Hageman factor cleavage product, HFf, were not distinguished in this assay and had a Km of 190 microM and kcat of 15/s. Treatment of HF with 10(-2) M diisopropylfluorophosphate yielded preparations containing 0.2 to 0.9% activated HF as assessed in plastic cuvettes. In quartz cuvettes, concave upward progress curves were obtained. Secondary plots of absorbance/time against time were also nonlinear and consistent with an increased rate of formation of activated enzyme. The curves varied with total protein content and synthetic substrate concentration in a manner consistent with autoactivation; increasing synthetic substrate competed with native HF for interaction with activated HF. Accelerated cleavage of surface-bound radiolabeled HF was demonstrated after incubation with HFa but not HFf, indicating that HFa is the form of active enzyme responsible for autocleavage. The autoactivation described herein may provide a sufficient initial concentration of activated HF to initiate the intrinsic coagulation, fibrinolytic, and kinin-forming cascade.
