Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary
Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for...
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Veröffentlicht in: | Journal of Chromatography A 2007-05, Vol.1150 (1), p.327-331 |
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description | Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20
mM glycine buffer (pH 8.6) whereas 20
mM β-alanine–hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200
nm. The whole study was performed on poorly soluble brominated substrate – 1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (
K
M) as well as the substrate inhibition constant (
K
SI). The value of
K
M and
K
SI obtained were 7.7
±
2.5
mM and 1.1
±
0.4
mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data. |
doi_str_mv | 10.1016/j.chroma.2006.09.022 |
format | Article |
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mM glycine buffer (pH 8.6) whereas 20
mM β-alanine–hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200
nm. The whole study was performed on poorly soluble brominated substrate – 1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (
K
M) as well as the substrate inhibition constant (
K
SI). The value of
K
M and
K
SI obtained were 7.7
±
2.5
mM and 1.1
±
0.4
mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.</description><identifier>ISSN: 0021-9673</identifier><identifier>DOI: 10.1016/j.chroma.2006.09.022</identifier><identifier>PMID: 17010980</identifier><identifier>CODEN: JOCRAM</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Electrophoresis, Capillary - methods ; EMMA ; Enzyme kinetics ; Enzymes and enzyme inhibitors ; Ethylene Dibromide - metabolism ; Fundamental and applied biological sciences. Psychology ; Haloalkane dehalogenase ; Hydrolases ; Hydrolases - antagonists & inhibitors ; Hydrolases - metabolism ; Kinetics ; Microchemistry - methods ; Sphingomonas - enzymology ; Substrate inhibition ; Substrate Specificity</subject><ispartof>Journal of Chromatography A, 2007-05, Vol.1150 (1), p.327-331</ispartof><rights>2006 Elsevier B.V.</rights><rights>2007 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c390t-b7a4d51956070203ccdaa1d19f5894926d33f2bea304d2d614636f98c818af363</citedby><cites>FETCH-LOGICAL-c390t-b7a4d51956070203ccdaa1d19f5894926d33f2bea304d2d614636f98c818af363</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.chroma.2006.09.022$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>309,310,314,780,784,789,790,3550,23930,23931,25140,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18794229$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17010980$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Papežová, Kateřina</creatorcontrib><creatorcontrib>Němec, Tomáš</creatorcontrib><creatorcontrib>Chaloupková, Radka</creatorcontrib><creatorcontrib>Glatz, Zdeněk</creatorcontrib><title>Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary</title><title>Journal of Chromatography A</title><addtitle>J Chromatogr A</addtitle><description>Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20
mM glycine buffer (pH 8.6) whereas 20
mM β-alanine–hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200
nm. The whole study was performed on poorly soluble brominated substrate – 1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (
K
M) as well as the substrate inhibition constant (
K
SI). The value of
K
M and
K
SI obtained were 7.7
±
2.5
mM and 1.1
±
0.4
mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Electrophoresis, Capillary - methods</subject><subject>EMMA</subject><subject>Enzyme kinetics</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Ethylene Dibromide - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Haloalkane dehalogenase</subject><subject>Hydrolases</subject><subject>Hydrolases - antagonists & inhibitors</subject><subject>Hydrolases - metabolism</subject><subject>Kinetics</subject><subject>Microchemistry - methods</subject><subject>Sphingomonas - enzymology</subject><subject>Substrate inhibition</subject><subject>Substrate Specificity</subject><issn>0021-9673</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1P3DAQhn2gAgr8A4RyaW-bju2sE18qVQgKElIPwNly_KH1KolT26mUf8_ArsStp5nD847eeQi5plBToOLHvja7FEddMwBRg6yBsRNyDsDoRoqWn5GvOe8BaAstOyVnOCnIDs5JeC6LXavoq7z0uSRdXBWmXehDCXGq-rVygzMlxXkXkyvB6GFYq9HZgKStxmBS1JMe1hwyBqtZpxI-GB-GAQmjZ1x0Wi_JF6-H7K6O84K83t-93D5snv78frz99bQxXELZ9K1u7JbKrYAWGHBjrNbUUum3nWwkE5Zzz3qnOTSWWUEbwYWXnelopz0X_IJ8P9ydU_y7uFzUGLJx2GFyccmqhUY0stsi2BxAfCHn5LyaUxixqaKg3rWqvTpoVe9aFUiFWjF2c7y_9OjhM3R0isC3I6Az6vJJTybkT65rZcOYRO7ngXNo419wSWUT3GTQbULlysbw_yZvYs6b7g</recordid><startdate>20070525</startdate><enddate>20070525</enddate><creator>Papežová, Kateřina</creator><creator>Němec, Tomáš</creator><creator>Chaloupková, Radka</creator><creator>Glatz, Zdeněk</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</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></search><sort><creationdate>20070525</creationdate><title>Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary</title><author>Papežová, Kateřina ; Němec, Tomáš ; Chaloupková, Radka ; Glatz, Zdeněk</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c390t-b7a4d51956070203ccdaa1d19f5894926d33f2bea304d2d614636f98c818af363</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Electrophoresis, Capillary - methods</topic><topic>EMMA</topic><topic>Enzyme kinetics</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Ethylene Dibromide - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Haloalkane dehalogenase</topic><topic>Hydrolases</topic><topic>Hydrolases - antagonists & inhibitors</topic><topic>Hydrolases - metabolism</topic><topic>Kinetics</topic><topic>Microchemistry - methods</topic><topic>Sphingomonas - enzymology</topic><topic>Substrate inhibition</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Papežová, Kateřina</creatorcontrib><creatorcontrib>Němec, Tomáš</creatorcontrib><creatorcontrib>Chaloupková, Radka</creatorcontrib><creatorcontrib>Glatz, Zdeněk</creatorcontrib><collection>Pascal-Francis</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><jtitle>Journal of Chromatography A</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Papežová, Kateřina</au><au>Němec, Tomáš</au><au>Chaloupková, Radka</au><au>Glatz, Zdeněk</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary</atitle><jtitle>Journal of Chromatography A</jtitle><addtitle>J Chromatogr A</addtitle><date>2007-05-25</date><risdate>2007</risdate><volume>1150</volume><issue>1</issue><spage>327</spage><epage>331</epage><pages>327-331</pages><issn>0021-9673</issn><coden>JOCRAM</coden><abstract>Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20
mM glycine buffer (pH 8.6) whereas 20
mM β-alanine–hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200
nm. The whole study was performed on poorly soluble brominated substrate – 1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (
K
M) as well as the substrate inhibition constant (
K
SI). The value of
K
M and
K
SI obtained were 7.7
±
2.5
mM and 1.1
±
0.4
mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>17010980</pmid><doi>10.1016/j.chroma.2006.09.022</doi><tpages>5</tpages></addata></record> |
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subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Electrophoresis, Capillary - methods EMMA Enzyme kinetics Enzymes and enzyme inhibitors Ethylene Dibromide - metabolism Fundamental and applied biological sciences. Psychology Haloalkane dehalogenase Hydrolases Hydrolases - antagonists & inhibitors Hydrolases - metabolism Kinetics Microchemistry - methods Sphingomonas - enzymology Substrate inhibition Substrate Specificity |
title | Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary |
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