This section describes a method of determining acid glucocerebrosidase (AGC) and neutral glucocerebrosidase (NGC) activities using fluorescent acceptor substrates and normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-ceramide (Cer) and C12-BODIPY-Cer, could be separated from the corresponding acceptor substrates, C6-NBD-glucosylceramide (GlcCer) and C12-BODIPY-GlcCer, within 5 min under the conditions used. |
Category | Glycolipids and related compounds |
Protocol Name | Assay of glucocerebrosidases using HPLC and fluorescent substrates |
Authors
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Hayashi, Yasuhiro
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Faculty of Pharmaceutical Sciences, Teikyo University
Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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C6-NBD-GlcCer (#N6783: Sigma-Aldrich, St. Louis, MO) |
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C12-BODIPY-GlcCer (#D7547: Invitrogen/Life Technologies, Carlsbad, CA) |
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Normal-phase column (Intersil SIL 15A-5: GL-Sciences, Tokyo, Japan) |
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Instruments
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HPLC (L-7100: Hitachi, Ltd., Tokyo, Japan) |
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Fluorescent detector (L-7480: Hitachi, Ltd., Tokyo, Japan) |
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Autosampler (L-7200: Hitachi, Ltd., Tokyo, Japan) |
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Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA) |
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Methods |
1. |
Assay of glucocerebrosidases using HPLC and fluorescent substrates
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1) |
For the AGC assay, 20 μL of reaction mixture should contain 0.6% sodium taurocholate, 0.25% Triton X-100, and 2.5 mM C6-NBD-GlcCer or 0.25 mM C12-BODIPY-GlcCer in 50 mM phosphate-citrate buffer, pH 5.0. For the NGC assay, 20 μL of reaction mixture should contain 0.25% sodium cholate, 1 mM CBE, and 2.5 mM C6-NBD-GlcCer in 50 mM Hepes buffer, pH 7.0. |
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2) |
Stop the reaction by adding 200 μL of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge the reaction mixture. |
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Dry the organic phase, dissolve lipids in 200 μL of isopropyl alcohol/n-hexane/H2O (55:44:1, v/v/v), and then transfer to a glass vial in an auto-sampler (HITACHI L-7200). |
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Determine fluorescence using a detector set to excitation and emission wavelengths of 470 and 530 nm for the assay with C6-NBD-GlcCer, and 505 and 540 nm for the assay with C12-BODIPY-GlcCer, respectively. |
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An aliquot of sample is automatically loaded onto a normal-phase column and eluted with isopropyl alcohol/n-hexane/H2O (55:44:1, v/v/v) for the assay using C6-NBD-GlcCer as a substrate or isopropyl alcohol/n-hexane/H2O (55:85:51, v/v/v) for that using C12-BODIPY-GlcCer as a substrate at a flow rate of 2.0 mL/min. |
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Notes | With this procedure, 50 fmol – 50 pmol of fluorescent Cer released from fluorescent GlcCer can be determined. |
Figure & Legends |
Figure & Legends
Reprinted from Anal Biochem., 383(1), Hayashi Y, Ito M. et al., A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases, 122-9, 2008, with permission from Elsevier. doi:10.1016/j.ab.2008.07.024. |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2013-11-27 14:35:59 |
- Yasuhiro Hayashi, Kota Zama, Eriko Abe, Nozomu Okino, Takehiko Inoue, Kousaku Ohno, and Makoto Ito. (2008) A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidase. Anal. Biochem. 383, 122-129 [PMID : 18708024]
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