Recombinant Human Hyaluronidase 2/HYAL2 His-tag Protein, CF

rhHYAL-2 recognizes repeating subunits of -GlcA-beta 1,­3-GlcNAc-beta 1,4- and hydrolyzes high molecular weight hyaluronic acid to intermediate and smaller sized products.

1 μg/lane of rhHYAL-2 (Catalog # 11012-GH) was resolved with SDS-PAGE under reducing (R) conditions and visualized by Silver Staining, showing a band at 55 kDa.

Lane 1 contained substrate Cy5-labeled LMW-HA GL401. In the presence of rhHYAL-2, the Hyaluronic Acid was modified to smaller molecular weight products with increasing amounts of rhHYAL-2 from left to right and a mobility shift was observed.

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

Recombinant Human Hyaluronidase 2/HYAL2 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to digest Cy5-Labeled Hyaluronan (Low MW) to dp1-dp4.
  The majority of Cy5-Labeled Hyaluronan (Low MW) (2 pmol) is digested by 0.25 μg of Recombinant Human HYAL-2, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Hyaluronidase 2/HYAL2 protein
  Met21-Gly447, with a C-terminal 6-His tag
• Accession #: Q12891.4
• N-terminal Sequence: Met21
• Protein/Peptide Type: Recombinant Enzymes
• 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 50 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: 53-58 kDa, under 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: 50 mM MES, pH 5.0
  • Recombinant Human HYAL-2 (rhHYAL-2) (Catalog # 11012-GH)
  • Cy5-Labeled Hyaluronan (Low MW) (Cy5 LMW-HA) (Catalog # GL401)
  • 17% SDS-PAGE gel and SDS-PAGE Reagents
  • Reducing SDS-PAGE sample buffer
  • Fluorescent imager
  1. Dilute rhHYAL-2 to 25 ng/µL in Assay Buffer.
  2. Dilute Cy5 LMW-HA to 0.2 µM in Assay Buffer.
  3. Prepare reaction by combining 10 µL of diluted rhHYAL-2 and 10 µL of diluted Cy5 LMW-HA.
  4. Prepare a negative control by combining 10 µL of diluted Cy5 LMW-HA and 10 µL of Assay Buffer.
  5. Incubate reaction(s) and negative control at room temperature for 18 hours.
  6. Add 7 µL of the reducing SDS-PAGE sample buffer to each reaction and negative control.
  7. Load 13.5 µL each sample and control per well on a 17% SDS-PAGE gel and run SDS-PAGE until the dye front has migrated more than two thirds of the way down the gel.
  8. Acquire gel image with a fluorescent imager.
  9. Visually determine the digestion of Cy5 LMW-HA by rhHYAL-2 to dp 1-4 in each reaction lane.
  Per Reaction:
  • rhHYAL-2: 0.25 µg
  • Cy5 LMW-HA: 2 pmol

Notes

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

Alternate Names for Recombinant Human Hyaluronidase 2/HYAL2 His-tag Protein, CF

• EC 3.2.1.35
• HYAL-2
• Hyaluronidase 2
• hyaluronoglucosaminidase 2
• hyaluronoglucosaminidase-2
• LUCA2
• LUCA-2
• LUCA2hyaluronidase-2
• Lung carcinoma protein 2
• lysosomal hyaluronidase
• PH20 homolog
• PH-20 homolog

Background

Human hyaluronidases (HYALs) are a group of five endo­-beta -­N­-acetyl­hexosaminidases that include HYAL1, HYAL2, HYAL3, HYAL4, and SPAM1 (PH20) (1, 2). While HYAL1, HYAL2 and SPAM1 are mainly active on hyaluronan (3, 4, 5), HYAL4 is active on both hyaluronan (6) and chondroitin sulfate (type C and D in particular) (7). Hyaluronan and chondroitin sulfate are abundant extracellular matrix components that have numerous biological functions (8). The backbone of chondroitin sulfate composed of repeating units of ­4-GlcA-beta 1,­3-GalNAc-beta 1­ most closely resembles to that of hyaluronan composed of repeating units of ­4-GlcA-beta 1,­3-GlcNAc-beta 1­. While HYAL1 is a strict lysosomal hyaluronidase and is active when pH ≤ 5.5 (9), HYAL2 is active in a narrow range of pH 5.0 to 5.5 (6) and is attached to cell membrane via GPI linker (10) where they may serve to hydrolyze high molecular weight hyaluronic acid of extracellular matrix to intermediate and small sized products.

1. Csoka, A.B. et al. (2001) Matrix Biol. 20:499.
2. Jedrzejas, M. J. and Stern, R. (2005) Proteins 61:227.
3. Csoka, A.B. et al. (1999) Genomics 60:351.
4. Lin, Y. et al. (1994) J. Cell Biol.125:1157.
5. Lepperdinger, G. et al. (2001) Matrix Biol. 20:509.
6. Wu, Z.L. and Ertelt, J.M. (2021) Glycobiology, cwab061, https://doi.org/10.1093/glycob/cwab061.
7. Kaneiwa, T. et al. (2010) Glycobiology 20:300.
8. Iozzo, R.V. (1998) Annu. Rev. Biochem. 67:609.
9. Hofinger, E.S.A. et al. (2007) Glycobiology 17:444.
10. Rai, S.K. et al. (2001). Proc. Natl. Acad. Sci. U.S.A, 98:4443.

Publications for HYAL2 (11012-GH)(1)

Publication using 11012-GH

• Narita, T;Tobisawa, Y;Bobkov, A;Jackson, M;Ohyama, C;Irie, F;Yamaguchi, Y; TMEM2 Is a Bona Fide Hyaluronidase Possessing Intrinsic Catalytic Activity The Journal of biological chemistry 2023-07-30 [PMID: 37527776] (Bioassay, Human)

Bioinformatics

• Uniprot:
  • Q12891.4()