Glia-Derived Nexin (GDN), also known as protease nexin-1 (PN-1), is a serine protease inhibitor secreted by glial cells. It belongs to the serpin superfamily and plays a crucial role in regulating extracellular proteolysis, particularly by inhibiting thrombin and plasminogen activators. GDN is involved in neurite outgrowth, synaptic plasticity, and tissue repair, making it important in nervous system development and neuroprotection. Studies suggest its role in preventing excessive proteolytic activity that could damage neural networks. The activity of recombinant human GDN was measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. Trypsin was diluted to 50 ug/ml in the assay buffer and 25 ul different concentrations of recombinant human GDN (MW: 45.7 KD) was incubated with 25 ul diluted trypsin at 37 ℃ for 30 minutes. Loading 50 µL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µL of 20 µM substrate. Include a substrate blank containing 50 µL of assay buffer and 50 µL of 20 µM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant human GDN significantly decreased trypsin activity. The inhibition IC50 was <42 nM.