• Purity

  >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH    ₂ (Catalog # ). The specific activity is >4,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ile24-Ser247, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ile24

• Structure / Form

  Active form

• Predicted Molecular Mass

  26 kDa

• SDS-PAGE

  20-24 kDa, non-reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)

• Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)

• Substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂(Catalog # ), 2 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhTrypsin 3 to 0.06 µg/mL in Assay Buffer.

2. Dilute Substrate to 20 µM in Assay Buffer.

3. Load 50 µL of 0.06 µg/mL of rhTrypsin 3 into 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.

4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.

5. Calculate specific activity: 

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhTrypsin 3 (Active form): 0.003 µg

• Substrate: 10 µM

Background: Trypsin 3/PRSS3

Human Trypsin 3, encoded by the PRSS3 gene, is also known as mesotrypsin (1). Constituting less than 10% of the total trypsin content in normal pancreatic juice, it is one of the three trypsin isoforms produced by the pancreas (2). Compared to trypsin 1 and 2, one intriguing feature of Trypsin 3 is its resistance to polypeptide trypsin inhibitors, such as the Kunitz-type soybean trypsin inhibitor or the Kazal-type pancreatic secretory trypsin inhibitor. As revealed by the crystal structure, this resistance is likely due to the presence of an arginine residue in place of the highly conserved Gly¹⁹⁸ (3). Trypsin 3 is synthesized in the pancreas and secreted into the duodenum lumen, where it is activated by enterokinase. One proposed physiological function of Trypsin 3 is degradation of trypsin inhibitors, which facilitates the digestion of those foods rich in these proteins (4).

• References:

1. Nyaruhucha, C. N. M. et al. (1997) J. Biol. Chem. 272:10573.

2. Rinderknecht, H. et al. (1984) Gastroenterology 86:681.

3. Katona, G. et al. (2002) J. Mol. Biol. 315:1209.

4. Szmola, R. et al. (2003) J. Biol. Chem. 278:48580.

• Entrez Gene IDs:

  5646 (Human); 22073 (Mouse); 362347 (Rat)

• Alternate Names:

  Brain trypsinogen; EC 3.4.21; EC 3.4.21.4; Mesotrypsin; mesotrypsinogen; MTG; pancreatic trypsinogen III; protease, serine, 3 (mesotrypsin); protease, serine, 3; protease, serine, 4 (trypsin 4, brain); PRSS3; PRSS4; Serine protease 3; Serine protease 4; T9; TRY3MTG; TRY4mesotrypsin; Trypsin 3; Trypsin 4; Trypsin III; Trypsin IV; trypsin-3; Trypsinogen 15; trypsinogen 4; trypsinogen 5; trypsinogen IV