Kininogen 1 (KNG1), specifically its high-molecular-weight form (HMWK), is a multifunctional plasma glycoprotein that plays a critical role in the contact activation system of intrinsic blood coagulation. It acts as a non-enzymatic cofactor, circulating in a complex with prekallikrein and factor XI. Upon binding to negatively charged surfaces, HMWK facilitates the activation of these factors, leading to the initiation of coagulation and the inflammatory kallikrein-kinin system.The activity of Kininogen 1 (KNG1) was measured by its ability to inhibit papain cleavage of the fluorogenic peptide substrate Z-Phe-Arg-AMC in assay buffer (50 mM Tris, pH 7.0). Papain was diluted to 100 μg/mL in activation buffer (50 mM Tris, 5 mM DTT, pH 7.0) and allowed to stand at room temperature for 10 minutes, then further diluted to 2 μg/mL with assay buffer. KNG1 was serially diluted 2-fold with assay buffer. Subsequently, 50 μL of diluted papain (2 μg/mL) was incubated with 50 μL of KNG1 at room temperature for 10 minutes. The incubated mixture was diluted 1:5 with assay buffer, and 50 μL of this diluted mixture was mixed with 50 μL of Z-Phe-Arg-AMC substrate (200 μM, prepared by dissolving in DMSO to 10 mM and then diluting with assay buffer). A substrate blank containing 50 μL of assay buffer and 50 μL of substrate was also included. Reading was performed in kinetic mode at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes. The results showed that KNG1 significantly inhibited papain activity.The result was shown in Figure 1 and it was obvious that recombinant mouse KNG1 significantly decreased trypsin activity. The inhibition IC50 was <39 nM.