Active Alpha-1-Microglobulin (a1M)

Alpha-1-Microglobulin (a1M) is a small protein belonging to the superfamily of lipocalins, which is present in various body fluids such as plasma and cerebrospinal fluid. it has a role in the transportation of molecules like metals and serves as a marker for certain diseases, including kidney and liver disorders.The activity of recombinant human A1M was measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(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 a1M (MW: 64 KD) was incubated with 25ul diluted trypsin at 37 ℃ for 15 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.nclude 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 a1M significantly decreased trypsin activity. The inhibition IC50 was <2.2 nM.