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A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities
The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of I 3 - has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometr...
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| Published in: | Analytical biochemistry 2008-08, Vol.379 (1), p.60-65 |
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| Main Authors: | , , , , , |
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| cites | cdi_FETCH-LOGICAL-c348t-c4c85927bd024233a1da0adb4ddc98d02d3c6762402ef86f93358ece5ada32b3 |
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| container_title | Analytical biochemistry |
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| creator | Verhaeghe, Elodie Buisson, David Zekri, Elisabeth Leblanc, Catherine Potin, Philippe Ambroise, Yves |
| description | The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of
I
3
-
has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H
2O
2/I
− catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (
K
m and
k
cat) of bromide and iodide for vanadium bromoperoxidase from
Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard
I
3
-
assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases. |
| doi_str_mv | 10.1016/j.ab.2008.04.041 |
| format | article |
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I
3
-
has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H
2O
2/I
− catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (
K
m and
k
cat) of bromide and iodide for vanadium bromoperoxidase from
Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard
I
3
-
assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2008.04.041</identifier><identifier>PMID: 18492479</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Algal Proteins - metabolism ; Ascophyllum - enzymology ; Biological Assay - methods ; Bromoperoxidase ; Chromatography, High Pressure Liquid ; Colorimetry - methods ; Hydrogen-Ion Concentration ; Iodide Peroxidase - metabolism ; Iodoperoxidase ; Kinetics ; Mass Spectrometry ; Peroxidases - metabolism ; Thymol blue ; Thymolphthalein - analogs & derivatives ; Thymolphthalein - chemistry ; Thymolphthalein - metabolism ; Vanadium ; Vanadium - chemistry ; Vanadium - metabolism</subject><ispartof>Analytical biochemistry, 2008-08, Vol.379 (1), p.60-65</ispartof><rights>2008 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c348t-c4c85927bd024233a1da0adb4ddc98d02d3c6762402ef86f93358ece5ada32b3</citedby><cites>FETCH-LOGICAL-c348t-c4c85927bd024233a1da0adb4ddc98d02d3c6762402ef86f93358ece5ada32b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18492479$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Verhaeghe, Elodie</creatorcontrib><creatorcontrib>Buisson, David</creatorcontrib><creatorcontrib>Zekri, Elisabeth</creatorcontrib><creatorcontrib>Leblanc, Catherine</creatorcontrib><creatorcontrib>Potin, Philippe</creatorcontrib><creatorcontrib>Ambroise, Yves</creatorcontrib><title>A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of
I
3
-
has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H
2O
2/I
− catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (
K
m and
k
cat) of bromide and iodide for vanadium bromoperoxidase from
Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard
I
3
-
assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.</description><subject>Algal Proteins - metabolism</subject><subject>Ascophyllum - enzymology</subject><subject>Biological Assay - methods</subject><subject>Bromoperoxidase</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Colorimetry - methods</subject><subject>Hydrogen-Ion Concentration</subject><subject>Iodide Peroxidase - metabolism</subject><subject>Iodoperoxidase</subject><subject>Kinetics</subject><subject>Mass Spectrometry</subject><subject>Peroxidases - metabolism</subject><subject>Thymol blue</subject><subject>Thymolphthalein - analogs & derivatives</subject><subject>Thymolphthalein - chemistry</subject><subject>Thymolphthalein - metabolism</subject><subject>Vanadium</subject><subject>Vanadium - chemistry</subject><subject>Vanadium - metabolism</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><recordid>eNp1kM1rGzEQxUVoSNwk95zKnnpbd_Th3VVvIbRJIdBLyFXMSrMgs7YcjRzi_z4KNvRUeDAw_N6D94S4lbCUILsf6yWOSwUwLMFUyTOxkGC7FjTYL2IBALpVne0vxVfmNYCUZtVdiEs5GKtMbxfi5a7xaU45bqjk6BtkxkMzpdxwIQyHlgsWanCL84GJmzQ1MYXU1k9oxpw2aUc5vceAXClf4lsskfhanE84M92c7pV4_v3r-f6xffr78Of-7qn12gyl9cYPK6v6MYAySmuUAQHDaELwdqjPoH3Xd8qAomnoJqv1aiBPKwyo1aivxPdj7C6n1z1xcZvInuYZt5T27DqrZC97VUE4gj4n5kyT29XKmA9Ogvuc0q0dju5zSgemSlbLt1P2ftxQ-Gc4bVeBn0eAasG3SNmxj7T1FGImX1xI8f_pH69mhFA</recordid><startdate>20080801</startdate><enddate>20080801</enddate><creator>Verhaeghe, Elodie</creator><creator>Buisson, David</creator><creator>Zekri, Elisabeth</creator><creator>Leblanc, Catherine</creator><creator>Potin, Philippe</creator><creator>Ambroise, Yves</creator><general>Elsevier Inc</general><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></search><sort><creationdate>20080801</creationdate><title>A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities</title><author>Verhaeghe, Elodie ; Buisson, David ; Zekri, Elisabeth ; Leblanc, Catherine ; Potin, Philippe ; Ambroise, Yves</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c348t-c4c85927bd024233a1da0adb4ddc98d02d3c6762402ef86f93358ece5ada32b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Algal Proteins - metabolism</topic><topic>Ascophyllum - enzymology</topic><topic>Biological Assay - methods</topic><topic>Bromoperoxidase</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Colorimetry - methods</topic><topic>Hydrogen-Ion Concentration</topic><topic>Iodide Peroxidase - metabolism</topic><topic>Iodoperoxidase</topic><topic>Kinetics</topic><topic>Mass Spectrometry</topic><topic>Peroxidases - metabolism</topic><topic>Thymol blue</topic><topic>Thymolphthalein - analogs & derivatives</topic><topic>Thymolphthalein - chemistry</topic><topic>Thymolphthalein - metabolism</topic><topic>Vanadium</topic><topic>Vanadium - chemistry</topic><topic>Vanadium - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Verhaeghe, Elodie</creatorcontrib><creatorcontrib>Buisson, David</creatorcontrib><creatorcontrib>Zekri, Elisabeth</creatorcontrib><creatorcontrib>Leblanc, Catherine</creatorcontrib><creatorcontrib>Potin, Philippe</creatorcontrib><creatorcontrib>Ambroise, Yves</creatorcontrib><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><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Verhaeghe, Elodie</au><au>Buisson, David</au><au>Zekri, Elisabeth</au><au>Leblanc, Catherine</au><au>Potin, Philippe</au><au>Ambroise, Yves</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2008-08-01</date><risdate>2008</risdate><volume>379</volume><issue>1</issue><spage>60</spage><epage>65</epage><pages>60-65</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of
I
3
-
has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H
2O
2/I
− catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (
K
m and
k
cat) of bromide and iodide for vanadium bromoperoxidase from
Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard
I
3
-
assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>18492479</pmid><doi>10.1016/j.ab.2008.04.041</doi><tpages>6</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 2008-08, Vol.379 (1), p.60-65 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_69217172 |
| source | ScienceDirect Journals |
| subjects | Algal Proteins - metabolism Ascophyllum - enzymology Biological Assay - methods Bromoperoxidase Chromatography, High Pressure Liquid Colorimetry - methods Hydrogen-Ion Concentration Iodide Peroxidase - metabolism Iodoperoxidase Kinetics Mass Spectrometry Peroxidases - metabolism Thymol blue Thymolphthalein - analogs & derivatives Thymolphthalein - chemistry Thymolphthalein - metabolism Vanadium Vanadium - chemistry Vanadium - metabolism |
| title | A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities |
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