Analysis of endo-β-N-acetylglucosaminidase activity by high-pressure liquid chromatography on a silica-based chemically bonded octadecyl column
✍ Scribed by Hitoo Iwase; Tetuo Morinaga; Yu-Teh Li; Su-Chen Li
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 235 KB
- Volume
- 113
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
We have devised a new method for assaying the endo-p-N-acetylglucosaminidase activity by using the dansyl asparaginyl oligosaccharide, (Man),(GlcNAc)z-Asn-DNS, as the substrate and analyzing the product, GlcNAc-Asn-DNS, by a reverse-phase high-pressure liquid chromatography using a silica-based chemically bonded octadecyl column (Waters PBondapack C,,). The column is eluted with 8% acetonitrile in 25 mM sodium borate buffer, pH 7.5, at 3 ml/min. The effluent is monitored by a Perkin-Elmer LC-75 uv monitor at 213 nm and a Perkin-Elmer LC-1000 fluorescence monitor (excitation, 313 nm; emission, 540 nm). Under these conditions, GlcNAc-Asn-DNS is well separated from (Man),(GlcNAc),-Asn-DNS and the analysis can be completed in 5 min. The peak height is used to quantify the dansyl derivatives. Under the conditions described above. the lower limit of detection is 0.1 nmol of dansyl glycopeptides.