• Purity

  >95%, 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 Val16-Ser247, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Val16

• Structure / Form

  Pro form

• Predicted Molecular Mass

  27 kDa

• SDS-PAGE

  32 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0 

• Recombinant Human Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3710-SE)

• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # )

• Bacterial Thermolysin (Thermolysin) (Catalog # )

• Substrate: Mca-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH₂ (Catalog # )

• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M in DMSO

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

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

1. Activate rhEnterokinase with Thermolysin.

1. Dilute rhEnterokinase to 100 µg/mL with Activation Buffer.

2. Dilute Thermolysin to 3.16 μg/mL with Activation Buffer. 

3. Mix equal volumes of diluted rhEnterokinase with Thermolysin

4. Incubate at 37 °C for 30 minutes.

5. Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline.

2. Activate rhTrypsin 3, with activated rhEnterokinase.

1. Dilute the activated rhEnterokinase to 2 µg/mL in Assay Buffer.

2. Dilute rhTrypsin 3 to 200 µg/mL in Assay Buffer.

3. Mix equal volumes of the diluted rhEnterokinase and diluted rhTrypsin 3.

4. Incubate at 37 °C for 90 minutes.

3. After incubation, dilute activated rhTrypsin 3 to 0.1 µg/mL in Assay Buffer.

4. Dilute Substrate to 20 µM in Assay Buffer.

5. Load in a black well plate 50 µL of rhTrypsin 3 at 0.1 µg/mL, 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.

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

7. Calculate specific activity:

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

Per Well:

• rhTrypsin 3: 0.005 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 physiologic function of Trypsin 3 has been proposed to be 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