• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• 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 >3,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala16-Ser247, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala16

• Structure / Form

  Pro form

• Predicted Molecular Mass

  26 kDa

• SDS-PAGE

  32 kDa and 36 kDa, reducing conditions

Assay Procedure

Materials

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

• Assay Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 8.0

• Recombinant Human Trypsin 1/PRSS1 (rhTrypsin 1) (Catalog # 3848-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 # ) 2 mM stock in DMSO

• 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 (see also R&D Systems, Catalog # 1585-SE).

1. Activate rhEnterokinase at 50 µg/mL with 1.58 µg/mL Thermolysin in Activation Buffer.

2. Incubate at 37 °C for 30 minutes.

3. Stop the reaction with an equal volume of 20 mM of 1,10 Phenanthroline for a final concentration of 10 mM.

2. Activate rhTrypsin 1 at 100 µg/mL with activated rhEnterokinase at 0.4 µg/mL.

1. Dilute the activated rhEnterokinase to 0.8 µg/mL in Assay Buffer.

2. Dilute rhTrypsin 1 to 200 µg/mL in Assay Buffer.

3. Mix equal volumes of 0.8 µg/mL rhEnterokinase with 200 µg/mL rhTrypsin 1.

4. Incubate reaction at room temperature for 15 minutes.

3. Dilute activated rhTrypsin 1 to 0.1 µg/mL in Assay Buffer.

4. Dilute Substrate to 20 µM in Assay Buffer.

5. In a plate load 50 µL of 0.1 µg/mL rhTrypsin 1 to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL 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 1: 0.005 µg

• Substrate: 10 µM

Background: Trypsin 1/PRSS1

Human Trypsin 1, encoded by the PRSS1 gene, is also known as cationic trypsinogen (1). Constituting approximately two-thirds of the total trypsin content in normal pancreatic juice, it is the most abundant trypsin isoform produced by the pancreas. It contains a signal peptide (residues 1‑15), a pro region (residues 16‑23), and a mature chain (residues 24‑247). Trypsin 1 is synthesized in the pancreas and secreted into the duodenum lumen, where it is activated by enterokinase. Its major physiologic function is to digest food and to activate other pro-enzymes (2). Mutations in the PRSS1 gene can cause hereditary pancreatitis (3).

• References:

1. Emi, M. et al. (1986) Gene 41:305.

2. Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1483, Academic Press, San Diego.

3. Teich, N. et al. (2006) Hum. Mutat. 27:721.

• Entrez Gene IDs:

  5644 (Human); 114228 (Mouse); 24691 (Rat)

• Alternate Names:

  Beta-trypsin; Cationic trypsinogen; digestive zymogen; DKFZp779A0837; EC 3.4.21; EC 3.4.21.4; FLJ57296; MGC120175; MGC149362; nonfunctional trypsin 1; protease, serine, 1 (trypsin 1); PRSS1; PTRYI; Serine protease 1; TRP1; TRY1beta-trypsin; TRY4; Trygn16; TRYP1; Trypsin 1; Trypsin I; trypsin-1; Trypsinogen 1; trypsinogen A