• 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 4-Methylumbelliferyl-beta -D-N,N’,N”-triacetylchitotriose (4-MU-TACT). The specific activity is >400 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ala22-Ser464 (major) & Lys23-Ser464 (minor), both with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala22 & Lys23

• Predicted Molecular Mass

  50 kDa

• SDS-PAGE

  54-57 kDa doublet, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 10 mM MES, 0.05% (w/v) Brij-35, pH 6.0

• Reading Buffer: 100 mM Tris, pH 9.0

• Recombinant Mouse Chitotriosidase/CHIT1 (rmCHIT1) (Catalog # 5325-GH)

• Substrate: 4-Methylumbelliferyl beta -D-N’,N”-triacetylchitotrioside (4-MU-TACT) (Sigma, Catalog # M-5639), 5 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 rmCHIT1 to 2 µg/mL in Assay Buffer.

2. Dilute Substrate to 200 µM in Assay Buffer.

3. Mix 20 µL of 2 µg/mL of rmCHIT1 and 20 µL of 200 µM of Substrate. Include a Substrate Blank containing 20 µL Assay Buffer and 20 µL Substrate.

4. Incubate at room temperature for 10 minutes. Protect from light.

5. Load 90 µL of Reading Buffer per sample into a plate.

6. Add 10 µL from each reaction mixture into wells containing Reading Buffer.

7. Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in endpoint mode.

8. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

Per Well:

• rmCHIT1: 0.01 µg

• Substrate: 10 µM

Background: Chitotriosidase/CHIT1

Chitotriosidase, encoded by the mouse CHIT1 gene, is a typical member of the chitinase family (1). It is distinct from another member known as acidic mammalian chitinase (AMC), encoded by the CHIA gene, in several aspects. AMC/CHIA is expressed mainly in alveolar macrophages and in both the mouse and human gastrointestinal tract (2). CHIT1, however, is expressed exclusively by phagocytes in humans and in the gastrointestinal tract, tongue, fore-stomach, and Paneth cells of the small intestine in mice (2). Both CHIA and CHIT1 are secreted as 50 kDa proteins. In contrast to CHIA, CHIT1 is not stable under acidic pH and can be processed into a C-terminally truncated 39 kDa form (2‑4). Human CHIT1 is the best of three biomarkers in the monitoring of Gaucher disease (5). The other two commonly used markers include acid phosphatase and angiotensin-converting enzyme (ACE) (5). Human CHIT1 is also a specific marker of macrophage activation in acute ischemic stroke (6).

• References:

1. Zheng, T. et al. (2005) Gene 357:37.

2. Boot, R.G. et al. (2006) J. Histochem. Cytochem. 53:1283.

3. Boot, R.G. et al. (2001) J. Biol. Chem. 276:6770.

4. Renkema, G.H. et al. (1997) Eur. J. Biochem. 244:279.

5. Vellodi, A. et al. (2005) J. Inherit. Metab. Dis. 28:585.

6. Sotgiu, S. et al. (2005) Eur. Neurol. 54:149.

• Entrez Gene IDs:

  1118 (Human)

• Alternate Names:

  CHI3; CHIT1; CHITCHI3; chitinase 1 (chitotriosidase); chitinase-1; Chitotriosidase; chitotriosidase-1; EC 3.2.1.14; FLJ00314; MGC125322; plasma methylumbelliferyl tetra-N-acetylchitotetraoside hydrolase