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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Assay of glucocerebrosidases using high-performance liquid chromatography and fluorescent substrates" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/05/11" /><meta name="citation_author" content="Yasuhiro Hayashi" /><meta name="citation_pmid" content="37590631" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593887/" /><meta name="citation_keywords" content="neutral glucocerebrosidase (NGC)" /><meta name="citation_keywords" content="fluorescent substrates" /><meta name="citation_keywords" content="acid glucocerebrosidase (AGC)" /><meta name="citation_keywords" content="high-performance liquid chromatography (HPLC)" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Assay of glucocerebrosidases using high-performance liquid chromatography and fluorescent substrates" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Yasuhiro Hayashi" /><meta name="DC.Date" content="2022/05/11" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593887/" /><meta name="description" content="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." /><meta name="og:title" content="Assay of glucocerebrosidases using high-performance liquid chromatography and fluorescent substrates" /><meta name="og:type" content="book" /><meta name="og:description" content="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." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593887/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-glycopodv2-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/glycopodv2/g151-glucocerebros/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593887/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} </style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script>
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593887_"><span class="title" itemprop="name">Assay of glucocerebrosidases using high-performance liquid chromatography and fluorescent substrates</span></h1><div class="contrib half_rhythm"><span itemprop="author">Yasuhiro Hayashi</span>, Ph.D.<div class="affiliation small">Faculty of Agriculture, University of Miyazaki<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ikazayim.cc@orihusay_ihsayah" class="oemail">pj.ca.u-ikazayim.cc@orihusay_ihsayah</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 6, 2021</span>; Last Revision: <span itemprop="dateModified">May 11, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g151-glucocerebros.Introduction"><h2 id="_g151-glucocerebros_Introduction_">Introduction</h2><p>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.</p></div><div id="g151-glucocerebros.Protocol"><h2 id="_g151-glucocerebros_Protocol_">Protocol</h2><p>In this chapter, the protocol for glucocerebrosidase assay using HPLC and fluorescent substrates will be described (<b>Note 1</b>).</p><div id="g151-glucocerebros.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">C6-NBD-GlcCer (#N6783: Sigma-Aldrich, St. Louis, MO)</p></dd><dt>2.</dt><dd><p class="no_top_margin">C12-BODIPY-GlcCer (#D7547: Invitrogen/Life Technologies, Carlsbad, CA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Normal-phase column (Intersil SIL 15A-5: GL-Sciences, Tokyo, Japan)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Isopropyl alcohol</p></dd><dt>5.</dt><dd><p class="no_top_margin"><i>n</i>-hexane</p></dd></dl></div><div id="g151-glucocerebros.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">HPLC (L-7100: Hitachi, Ltd., Tokyo, Japan)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Fluorescent detector (L-7480: Hitachi, Ltd.)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Autosampler (L-7200: Hitachi, Ltd.)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)</p></dd></dl></div><div id="g151-glucocerebros.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Glucocerebrosidase assay using HPLC and fluorescent substrates</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">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.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Stop the reaction by adding 200 μL of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge the reaction mixture.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Dry the organic phase, dissolve lipids in 200 μL of isopropyl alcohol/n-hexane/H<sub>2</sub>O (55:44:1, v/v/v), and then transfer to a glass vial in an autosampler (Hitachi, Ltd.: L-7200).</p></dd><dt>d.</dt><dd><p class="no_top_margin">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.</p></dd><dt>e.</dt><dd><p class="no_top_margin">An aliquot of sample is automatically loaded onto a normal-phase column and eluted with isopropyl alcohol/<i>n</i>-hexane/H<sub>2</sub>O (55:44:1, v/v/v) for the assay using C6-NBD-GlcCer as a substrate or isopropyl alcohol/n-hexane/H<sub>2</sub>O (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.</p></dd></dl></dd></dl></div><div id="g151-glucocerebros.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">With this procedure, 50 fmol–50 pmol of fluorescent Cer released from fluorescent GlcCer can be determined.</p></dd></dl><div id="g151-glucocerebros.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593887/bin/g151-glucocerebros-Image001.jpg" alt="Figure 1: . Detection of fluorescence-labeled Cer and GlcCer analogs on normal-phase high-performance liquid chromatography (HPLC)." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>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/<i>n</i>-hexane/H<sub>2</sub>O (55:44:1) (A) or isopropyl alcohol/<i>n</i>-hexane/H<sub>2</sub>O (55:85:1) (B) at a flow rate of 2 mL/min at room temperature.</p></div><div class="permissions">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.</div></div></div></div><div id="g151-glucocerebros.References"><h2 id="_g151-glucocerebros_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g151-glucocerebros.REF.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. <span><span class="ref-journal">Anal Biochem. </span>2008;<span class="ref-vol">383</span>:122–129.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18708024" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18708024</span></a>] [<a href="http://dx.crossref.org/10.1016/j.ab.2008.07.024" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593887_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g151-glucocerebros.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div id="bk_toc_contnr"></div></div></div>
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