• 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 >1,500 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ala22-Asn466, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala22

• Predicted Molecular Mass

  51 kDa

• SDS-PAGE

  52 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 10 mM MES, pH 6.0

• Reading Buffer: 100 mM Tris, pH 9.0

• Recombinant Human Chitotriosidase/CHIT1 (rhCHIT1) (Catalog # 3559-GH)

• Substrate: 4-Methylumbelliferyl beta -D-N,N’,N”-triacetylchitotriose (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 rhCHIT1 to 0.4 ng/μL in Assay Buffer.

2. Dilute Substrate to 200 μM in Assay Buffer.

3. Mix equal volumes of 0.4 ng/μL rhCHIT1 and 200 μM Substrate. Include a Substrate Blank with equal volumes of Assay Buffer and Substrate.

4. Incubate at room temperature for 5 minutes. Protect from light.

5. Load 90 μL of Reading Buffer per sample in plate.

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

7. Immediately 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:

• rhCHIT1: 0.002 μg

• Substrate: 10 μM

Background: Chitotriosidase/CHIT1

Chitotriosidase encoded by the CHIT1 gene is a member of the chitinase family that is selectively expressed in activated tissue macrophages (1). It is distinct from another member, known as acidic mammalian chitinase (AMC) encoded by the CHIA gene and expressed mainly in the gastrointestinal tract and lung (2). Both CHIA and CHIT1 are secreted as 50 kDa proteins. In contrast to CHIA, CHIT1 is not stable under acidic pHs and can be processed into a C-terminally truncated 39 kDa form (2, 3). CHIT1 is the best biomarker in the monitoring of Gaucher disease among the three most commonly used markers that also include acid phosphatase and angiotensin-converting enzyme (ACE) (4). CHIT1 is also a specific marker of macrophage activation in acute ischemic stroke (5).

• References:

1. Aguilera, B. et al. (2003) J. Biol. Chem. 278:40911.

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

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

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

5. 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