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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
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 in our method.
Protocol
In this chapter, the protocol for glucocerebrosidase assay using HPLC and fluorescent substrates will be described (Note 1).
Materials
- 1.
C6-NBD-GlcCer (#N6783: Sigma-Aldrich, St. Louis, MO)
- 2.
C12-BODIPY-GlcCer (#D7547: Invitrogen/Life Technologies, Carlsbad, CA)
- 3.
Normal-phase column (Intersil SIL 15A-5: GL-Sciences, Tokyo, Japan)
- 4.
Isopropyl alcohol
- 5.
n-hexane
Instruments
- 1.
HPLC (L-7100: Hitachi, Ltd., Tokyo, Japan)
- 2.
Fluorescent detector (L-7480: Hitachi, Ltd.)
- 3.
Autosampler (L-7200: Hitachi, Ltd.)
- 4.
Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
Methods
- 1.
Glucocerebrosidase assay using HPLC and fluorescent substrates
- a.
For the AGC assay, 20 μL of the reaction mixture should contain 0.6% sodium taurocholate, 0.25% Triton X-100, and 2.5 mM of C6-NBD-GlcCer or 0.25 mM of C12-BODIPY-GlcCer in 50 mM of phosphate-citrate buffer, pH 5.0. For the NGC assay, 20 μL of the reaction mixture should contain 0.25% sodium cholate, 1 mM of CBE, and 2.5 mM of C6-NBD-GlcCer in 50 mM of Hepes buffer, pH 7.0.
- b.
Stop the reaction by adding 200 μL of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge the reaction mixture.
- c.
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 autosampler (Hitachi, Ltd.: L-7200).
- d.
Determine fluorescence using a detector set at 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.
- e.
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 the assay using C12-BODIPY-GlcCer as a substrate at a flow rate of 2.0 mL/min.
Notes
- 1.
With this procedure, 50 fmol–50 pmol of fluorescent Cer released from fluorescent GlcCer can be determined.
Figure 1:
Detection of fluorescence-labeled Cer and GlcCer analogs on normal-phase high-performance liquid chromatography (HPLC). Profile of HPLC of (A) C6-NBD-Cer and GlcCer (50 picomol each) and (B) C12-BODIPY-Cer and GlcCer (5 picomol each). Substrates and products were separated on a normal-phase column (Inertsil SIL 150A-5) using HPLC. Fluorescence was detected using a fluorescent detector. Samples were eluted via HPLC with isopropyl alcohol/n-hexane/H2O (55:44:1) (A) or isopropyl alcohol/n-hexane/H2O (55:85:1) (B) at a flow rate of 2 mL/min at room temperature.
References
- 1.
- Hayashi Y, Zama K, Abe E, Okino N, Inoue T, Ohno K, Ito M. A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidase. Anal Biochem. 2008;383:122–129. [PubMed: 18708024] [CrossRef]
Footnotes
The authors declare no competing or financial interests.