Recombinant Human Hexosaminidase A/HEXA Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Hexosaminidase A/HEXA Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 4-methylumbelliferyl-N-acetyl-beta -D-glucosaminide (4-MU-GlcNAc) The specific activity is >1,250 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Hexosaminidase A/HEXA protein
  Met1-Thr529, with a C-terminal 6-His tag
• Accession #: P06865
• N-terminal Sequence: Leu23
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HEXA
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Theoretical MW: 59 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 57-61 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 100 mM Sodium Citrate, 250 mM NaCl, pH 4.5
  • Recombinant Human Hexosaminidase A/HEXA (rhHEXA) (Catalog # 6237-GH)
  • Substrate: 4-Methylumbelliferyl-N-Acetyl-beta -D-glucosaminide (4-MU-GlcNAc) (Sigma, Catalog # M2133), 50 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 rhHEXA to 5 ng/μL in Assay Buffer.
  2. Dilute Substrate to 800 μM in Assay Buffer.
  3. Load into a plate 50 μL of 5 ng/μL rhHEXA, and start the reaction by adding 50 μL of 800 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 800 μM Substrate.
  4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

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

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

  Per Well:
  • rhHEXA: 0.250 μg
  • Substrate: 400 μM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Hexosaminidase A/HEXA Protein, CF

• beta-hexosaminidase subunit alpha
• Beta-N-acetylhexosaminidase subunit alpha
• EC 3.2.1
• EC 3.2.1.52
• HEXA
• hexosaminidase A (alpha polypeptide)
• Hexosaminidase A
• Hexosaminidase subunit A
• MGC99608
• N-acetyl-beta-glucosaminidase subunit alpha
• TSD

Background

beta -hexosaminidases are enzymes involved in the hydrolysis of terminal N-acetyl-D-hexosamine residues in GM2 gangliosides and globo-sphingolipids in lysosomes
(1‑4). The enzymes are composed of two alpha and/or beta  subunits, which are coded by HEXA and HEXB genes, respectively. Different association of the alpha and beta subunits gives rise to beta ‑hexosaminidase isoforms A, B and S (Hex A, B and S) (5), which have the composition of alpha beta , beta beta and alpha alpha , respectively. Our recombinant HEXA is presumably isoform Hex S, because only alpha subunit was expressed. Hex S is suggested to releases non‑reducing end N-acetylgalactosamine residues from dermatan sulfate, chondroitin sulfate and sulfated glycolipid SM2 (6). Recombinant HEXA is also highly active on 4-methylumbelliferyl-N-acetyl-beta -D-glucosaminide (6). Mutations in HEXA and HEXB genes cause lysosomal lipid storage disorders. Specifically, mutations of HEXA cause Tay-Sachs disease, manifested by the harmful accumulation of ganglioside GM2 in tissues and nerve cells in the brain (7‑10). Children with this disease usually die by age 4.

1. Gilbert, F. et al. (1975) Proc. Natl. Acad. Sci. USA 72:263.
2. Myerowitz, R. et al. (1985) Proc. Natl. Acad. Sci. USA 82:7830.
3. Korneluk, R.G. et al. (1986) J. Biol. Chem. 261:8407.
4. Mark, B.L. et al. (2003) J. Mol. Biol. 327:1093.
5. Mahuran, D.J. et al. (1988) J. Biol. Chem. 263:4612.
6. Hepbildikler, S.T. et al. (2002) J. Biol. Chem. 277:2562.
7. Mahuran, D.J. (1991) Biochim. Biophys. Acta 1096:87.
8. Mencarelli, S. et al. (2005) FEBS Lett. 579:5501.
9. Neufeld, E.F. (1989) J. Biol. Chem. 264:10927.
10. Ohno, K. et al. (2008) Mol. Genet. Metab. 94:462.

Publications for Hexosaminidase A/HEXA (6237-GH)(4)

Publications using 6237-GH

• J Désiré, Q Foucart, A Poveda, G Gourlaouen, Y Shimadate, M Kise, C Proceviat, R Ashmus, DJ Vocadlo, J Jiménez-Ba, A Kato, Y Blériot Synthesis, conformational analysis and glycosidase inhibition of bicyclic nojirimycin C-glycosides based on an octahydrofuro[3,2-b]pyridine motif Carbohydrate research, 2021-12-20;511(0):108491. 2021-12-20 [PMID: 34953389] (Bioassay, Human)
• RJ Boado, JZ Lu, EK Hui, H Lin, WM Pardridge Bi-functional IgG-lysosomal enzyme fusion proteins for brain drug delivery Sci Rep, 2019-12-09;9(1):18632. 2019-12-09 [PMID: 31819150] (Bioassay, Human)
• FN Njume, SM Ghogomu, RA Shey, LOT Gainkam, P Poelvoorde, P Humblet, J Kamgno, A Robert, L Mutesa, C Lelubre, E Edelweiss, A Poterszman, S Anheuser, L Vanhamme, J Souopgui Identification and characterization of the Onchocerca volvulus Excretory Secretory Product Ov28CRP, a putative GM2 activator protein PLoS Negl Trop Dis, 2019-07-22;13(7):e0007591. 2019-07-22 [PMID: 31329585] (Bioassay, Nematode - Onchocerca volvulus)
• Thaysen-Andersen M, Venkatakrishnan V, Loke I, Laurini C, Diestel S, Parker B, Packer N Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum. J Biol Chem, 2015-02-02;290(14):8789-802. 2015-02-02 [PMID: 25645918] (Bioassay)

Bioinformatics

• Gene Symbol: HEXA
• Uniprot:
  • P06865()