Recombinant Human Chymase/CMA1 Protein, CF

Catalog #: 4099-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
4099-SE-010

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Recombinant Human Chymase/CMA1 Protein, CF Summary

Product Specifications

Purity

>90%, 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, SUC-Ala-Ala-Pro-Phe-AMC. The specific activity is >80 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Chymase/CMA1 protein
Met1-Asn247, with a C-terminal 10-His tag

Accession #

P23946

N-terminal Sequence
Analysis

Gly20

Structure / Form

Pro form

Predicted Molecular Mass

27 kDa

SDS-PAGE

38 kDa, reducing conditions

Product Datasheets

4099-SE

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

4099-SE

Formulation	Supplied as a 0.2 μm filtered solution in HEPES and NaCl.
Shipping	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Maturation Buffer: 50 mM MES, pH 5.5
Cathepsin Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
Assay Buffer: 20 mM Tris, 2 M KCl, 0.02% (v/v) Triton^® X-100, pH 9.0
Recombinant Human Chymase/CMA1 (rhChymase) (Catalog # 4099-SE)
Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
N-Ethylmaleimide (NEM) (Sigma, Catalog # E1271), 50 mM stock in deionized water
Substrate: SUC-Ala-Ala-Pro-Phe-AMC (Bachem, Catalog # I-1465), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhChymase to 20 µg/mL in Maturation Buffer.
Dilute rmCathepsin C to 20 µg/mL in Cathepsin Buffer.
Dilute Heparin to 1.5 mg/mL in deionized water.
Combine 15 µL of 20 µg/mL rhChymase and 15 µL of 20 µg/mL rmCathepsin C.
Add 1 µL of Heparin and mix well.
Incubate at room temperature for 1 hour.
Stop maturation by adding 1.98 µL of 50 mM stock of NEM for a final concentration of 3 mM.
Dilute rhChymase to 2 µg/mL in Assay Buffer.
Incubate at room temperature for 5 minutes.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of 2 µg/mL rhChymase in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. As a Substrate Blank combine 50 μL of Substrate with 50 μL of Assay Buffer.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate Specific Activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

rhChymase: 0.100 µg
Substrate: 100 µM

Reconstitution Calculator

Background: Chymase/CMA1

Chymases are a group of chymotrypsin-like serine proteases secreted by mast cells (1). They are synthesized as inactive precursors containing a 2-residue propeptide, which needs to be removed by dipeptidyl peptidase I/cathepsin C for the enzymatic activity (2). Human Chymase encoded by the CMA1 gene is known to be involved in hypertention and heart failure through its ability to convert angiotensin I (Ang I) to angiotensin II (Ang II), which plays a key role in the regulation of arterial pressure (3). In addition, it is also important in physiological and pathological conditions including inflammation, fibrosis and processing of cytokines (4). Therefore, designing a specific inhibitor for Chymase activity has been a pharmacologic strategy to develop therapeutic agents.

References

Caughey, G.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. ed. p. 1531, Academic Press, San Diego.
Murakami, M. et al. (1995) J. Biol. Chem. 270:2218.
Miyazaki, M. and S. Takai (2006) J. Pharmacol. Sci. 100:391.
Nakajima, M. and N. Naya (2002) Jpn. J. Pharmacol. 90:206.

Entrez Gene IDs

1215 (Human); 17228 (Mouse); 25627 (Rat)

Alternate Names

Alpha-chymase; chymase 1 preproprotein transcript E; chymase 1 preproprotein transcript I; chymase 1, mast cell; Chymase; chymase, heart; chymase, mast cell; CMA1; CYH; CYM; EC 3.4.21; EC 3.4.21.39; Mast cell protease I; MCP3P; Mcpt5; MCT1; MGC119890; MGC119891