β-N-Acetylglucosamine and β-N-acetylgalactosamine residues are common constituents of glycoproteins and glycolipids. Commonly, β-N-acetylhexosaminidase has both hydrolysis activities for β-linked GlcNAc and GalNAc. The name “β-N-acetylhexosaminidase” stemmed from this dual glycon specificity. However, some β-N-acetylhexosaminidases hardly act on oligosaccharides but on p-nitrophenyl substrates. On the other hand, there are some enzymes that cannot act on artificial substrates but on natural substrates. Therefore it is recommended that β-N-acetylhexosaminidase assay should be done using both of artificial and natural substrates.
β-N-Acetylhexosaminidases are widely distributed in bacteria, fungi, plants, and animals. Among them, the human lysosomal enzyme is one of the important enzymes relating to genetic disease of metabolism of glycosphingolipid. The β-N-acetylhexosaminidase cleaves both β-linked GlcNAc and GalNAc residues at non-reducing end of oligosaccharides and glycoconjugates, and there are three β-N-acetylhexosaminidase isozymes, A, B, and S, in lysosome (Korneluk et al). All isozymes hydrolyze β-GlcNAc linkages in both glycoproteins and oligosaccharides. On the other hand, only isozyme A can hydrolyze β-GalNAc linkage in a ganglioside GM2 in the presence of glycolipid activator proteins (Wu et al., 1994). |
| Category | Glycosidases & related proteins |
| Protocol Name | Enzyme assay for β-N-acetylhexosaminidase |
Authors
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Ashida, Hisashi
Faculty of Biology-Oriented Science and Technology, Kinki University
Yamamoto, Kenji
*
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University
*To whom correspondence should be addressed.
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| KeyWords |
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Reagents
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| ● |
Artificial substrates: 4-nitrophenyl N-acetyl-β-D-glucosaminide (pNP-β-GlcNAc, Sigma-Aldrichm, St. Louis, MO), 4-nitrophenyl N-acetyl-β-D-galactosaminide (pNP-β-GalNAc, Sigma-Aldrich) |
| ● |
Natural substrates: N-acetyllactosamine (GlcNAcβ1-4Gal, Sigma-Aldrich), N,N’-diacetylchitobiose (GlcNAcβ1-4GlcNAc, Sigma-Aldrich), GlcNAcβ1-2Man (Tronto Research Chemicals, Toronto, Ontario), lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc, prepared from lacto-N-neotetraose by β-galactosidase digestion) |
| ● |
Glycolipid substrates: ganglioside GM2 (GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1Cer), asialo-GM2 (GalNAcβ1-4Galβ1-4Glcβ1-1Cer) |
| ● |
4-Nitrophenol, N-acetylglucosamine and N-acetylgalactosamine as standard substance |
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Silica-gel 60 TLC plate (5553, Merck Millipore, Billerica, MA) |
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Aniline (Wako Pure Chemical Industries, Ltd., Osaka, Japan) |
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Diphenylamine (Nacalai Tesque, Inc., Kyoto, Japan) |
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Orcinol (Wako Pure Chemical Industries, Ltd.) |
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4-(Dimethylamino)benzaldehyde (Sigma-Aldrich, Co., St. Louis. MO) |
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Instruments
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Voltex mixer (Scientific Industries, Inc., Bohemia, New York) |
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Spectrophotometer (Beckman Coulter, Inc., Brea, CA) |
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Block incubator (100°C) or boiling water bath |
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| Methods |
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1. |
Enzyme activity assay using pNP-substrates |
| 1) |
Incubate 50 μL substrate solution (1–2 mM) at 37°C |
Comment 0
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| 2) |
Add 10 μL enzyme solution in a suitable buffer |
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| 3) |
Incubate at 37°C for appropriate period |
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| 4) |
Stop the reaction by adding 500 μL of 0.2 M sodium borate buffer (pH 10.0) |
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| 5) |
Measure absorbance at 400 nm using spectrophotometer |
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2. |
TLC analysis on oligosaccharide and ganglioside substrates |
| 1) |
Incubate substrate solution (0.5–2 mM) with enzyme at 37°C in a suitable buffer |
Comment 1
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| 2) |
Spot 10 μL of the reaction mixture onto a silica-gel TLC plate |
Comment 0
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| 3) |
Develop TLC plate in chromatochamber using 1-butanol:acetic acid:water=2:1:1 (for oligosaccharides) or chloroform:methanol:0.02% CaCl2=5:4:1 (for gangliosides) as solvent |
Comment 0
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| 5) |
Spray with diphenylamine-aniline reagent (100 mL acetone, 1 mL aniline, 1 g diphenylamine, 10 mL phosphoric acid; stock 4°C) (Anderson et al., 2000) or orcinol-H2SO4 reagent (0.1 g orcinol, 6 mL H2SO4, 44 mL water) |
Comment 0
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| 6) |
Put the sprayed TLC plate between glasses |
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| 7) |
Heat at 120–150°C for 3–10 min in oven |
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3. |
Determination of amount of N-acetylglucosamine and N-acetylgalactosamine released from substrate (Reissig et al., 1955) |
| 1) |
Incubate substrate solution (0.5–2 mM) with enzyme at 37°C in a suitable buffer |
Comment 0
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| 2) |
Take 100 μL reaction mixture into microtube |
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| 3) |
Add 20 μL borate solution (4.95 g boric acid, adjust pH 9.1 using 1 M KOH, and fill up to 100 mL by water) and incubate at 100°C for 3 min in block incubator |
Comment 0
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| 4) |
Cool down in water (approximately 10°C) |
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| 5) |
Add 600 μL DMAB solution (10 g 4-(dimethylamino)benzaldehyde, 12.5 mL conc. HCl, 87.5 mL glacial acetic acid; stock 4°C; dilute 10 times with glacial acetic acid just before use) |
Comment 0
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| 7) |
Measure absorbance at 585 nm using spectrophotometer |
Comment 0
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
| Date of registration：2014-07-29 16:37:01 |
- Korneluk, R.G., Mahuran, D.J., Neote, K., Klavins, M.H., O’Dowd, B.F., Tropak, M., Willard, H.F., Anderson, M.J., Lowden, J.A., and Gravel, R.A. (1986) Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease. J. Biol. Chem. 261, 8407–8413 [PMID : 3013851]
- Wu, Y.Y., Lockyer, J.M., Sugiyama, E., Pavlova, N.V., Li, Y.T., and Li, S.C. (1994) Expression and specificity of human GM2 activator protein. J. Biol. Chem. 269, 16276–16283 [PMID : 8206933]
- Anderson, K., Li, S.C., and Li, Y.T. (2000) Diphenylamine-aniline-phosphoric acid reagent, a versatile spray reagent for revealing glycoconjugates on thin-layer chromatography plates. Anal. Biochem. 287, 337–339 [PMID : 11112283]
- Reissig, J.L., Storminger, J.L., and Leloir, L.F. (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217, 959–966 [PMID : 13271455]
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