Sensitive fluorogenic substrate for alkaline phosphatase
Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhi...
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| Veröffentlicht in: | Analytical biochemistry 2011-11, Vol.418 (2), p.247-252 |
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| container_title | Analytical biochemistry |
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| creator | Levine, Michael N. Raines, Ronald T. |
| description | Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications. |
| doi_str_mv | 10.1016/j.ab.2011.07.021 |
| format | Article |
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The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2011.07.021</identifier><identifier>PMID: 21827735</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Alkaline phosphatase ; Alkaline Phosphatase - analysis ; Alkaline Phosphatase - metabolism ; Catalysis ; catalytic activity ; chemical bonding ; ELISA ; enzyme-linked immunosorbent assay ; Enzyme-Linked Immunosorbent Assay - methods ; fluorescence ; Fluorescent Dyes - chemistry ; Hydrogen-Ion Concentration ; Latent fluorophore ; phosphates ; Rhodamine ; Rhodamines - chemistry ; Sensitivity and Specificity ; Spectrometry, Fluorescence - methods ; Trimethyl lock</subject><ispartof>Analytical biochemistry, 2011-11, Vol.418 (2), p.247-252</ispartof><rights>2011 Elsevier Inc.</rights><rights>Copyright © 2011 Elsevier Inc. All rights reserved.</rights><rights>2011 Elsevier Inc. All rights reserved. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c470t-5451bbc0a752597b0d828e360574dfd7a7778842ad48319fdcf810bad52450643</citedby><cites>FETCH-LOGICAL-c470t-5451bbc0a752597b0d828e360574dfd7a7778842ad48319fdcf810bad52450643</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.ab.2011.07.021$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,315,782,786,887,3552,27931,27932,46002</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21827735$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Levine, Michael N.</creatorcontrib><creatorcontrib>Raines, Ronald T.</creatorcontrib><title>Sensitive fluorogenic substrate for alkaline phosphatase</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.</description><subject>Alkaline phosphatase</subject><subject>Alkaline Phosphatase - analysis</subject><subject>Alkaline Phosphatase - metabolism</subject><subject>Catalysis</subject><subject>catalytic activity</subject><subject>chemical bonding</subject><subject>ELISA</subject><subject>enzyme-linked immunosorbent assay</subject><subject>Enzyme-Linked Immunosorbent Assay - methods</subject><subject>fluorescence</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Hydrogen-Ion Concentration</subject><subject>Latent fluorophore</subject><subject>phosphates</subject><subject>Rhodamine</subject><subject>Rhodamines - chemistry</subject><subject>Sensitivity and Specificity</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Trimethyl lock</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1v1DAURS0EokNh31U7O1YJz1-x0wUSqqAgVWJRurYc-2XG00w8tZOR-Pd4NG3VLlhZss-79-mYkDMKNQXafNnUtqsZUFqDqoHRN2RBoW0q4NC-JQsA4BVrWnVCPuS8gQIK2bwnJ4xqphSXC6JvccxhCntc9sMcU1zhGNwyz12ekp3KbUxLO9zbIYy43K1j3q3tZDN-JO96O2T89Hiekrsf3_9c_axufl__uvp2UzmhYKqkkLTrHFglmWxVB14zjbwBqYTvvbJKKa0Fs15oTtveu15T6KyXTEhoBD8lX4-5u7nbonc4lr0Gs0tha9NfE20wr1_GsDaruDecKsZbXgI-Pwak-DBjnsw2ZIfDYEeMczZat0LyVh6q4Ei6FHNO2D-3UDAH32ZjbGcOvg0oU3yXkfOX2z0PPAkuwMUR6G00dpVCNne3JaEpfwOa8UPr5ZHAYnEfMJnsAo4OfUjoJuNj-H__Pw-imTM</recordid><startdate>20111115</startdate><enddate>20111115</enddate><creator>Levine, Michael N.</creator><creator>Raines, Ronald T.</creator><general>Elsevier Inc</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20111115</creationdate><title>Sensitive fluorogenic substrate for alkaline phosphatase</title><author>Levine, Michael N. ; Raines, Ronald T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c470t-5451bbc0a752597b0d828e360574dfd7a7778842ad48319fdcf810bad52450643</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Alkaline phosphatase</topic><topic>Alkaline Phosphatase - analysis</topic><topic>Alkaline Phosphatase - metabolism</topic><topic>Catalysis</topic><topic>catalytic activity</topic><topic>chemical bonding</topic><topic>ELISA</topic><topic>enzyme-linked immunosorbent assay</topic><topic>Enzyme-Linked Immunosorbent Assay - methods</topic><topic>fluorescence</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Hydrogen-Ion Concentration</topic><topic>Latent fluorophore</topic><topic>phosphates</topic><topic>Rhodamine</topic><topic>Rhodamines - chemistry</topic><topic>Sensitivity and Specificity</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Trimethyl lock</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Levine, Michael N.</creatorcontrib><creatorcontrib>Raines, Ronald T.</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Levine, Michael N.</au><au>Raines, Ronald T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Sensitive fluorogenic substrate for alkaline phosphatase</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2011-11-15</date><risdate>2011</risdate><volume>418</volume><issue>2</issue><spage>247</spage><epage>252</epage><pages>247-252</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>21827735</pmid><doi>10.1016/j.ab.2011.07.021</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Analytical biochemistry, 2011-11, Vol.418 (2), p.247-252 |
| issn | 0003-2697 1096-0309 |
| language | eng |
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| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Alkaline phosphatase Alkaline Phosphatase - analysis Alkaline Phosphatase - metabolism Catalysis catalytic activity chemical bonding ELISA enzyme-linked immunosorbent assay Enzyme-Linked Immunosorbent Assay - methods fluorescence Fluorescent Dyes - chemistry Hydrogen-Ion Concentration Latent fluorophore phosphates Rhodamine Rhodamines - chemistry Sensitivity and Specificity Spectrometry, Fluorescence - methods Trimethyl lock |
| title | Sensitive fluorogenic substrate for alkaline phosphatase |
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