New support for the affinity chromatography of hemoglobin
Identifieur interne : 000B52 ( Istex/Corpus ); précédent : 000B51; suivant : 000B53New support for the affinity chromatography of hemoglobin
Auteurs : T. Görner ; P. Gissinger ; M. Léonard ; E. DellacherieSource :
- Journal of Chromatography B: Biomedical Sciences and Applications [ 0378-4347 ] ; 1997.
English descriptors
- KwdEn :
- Active immobilised, Allosteric, Allosteric site, Amino acids, Association constants, Benzenehexacarboxylic acid, Benzenetetracarboxylic acid, Binding constants, Central cavity, Chromatogr, Chromatographic support, Clinical aspects, Column ligands, Deoxyhb, Deoxyhemoglobin, Deoxyhemoglobin form, Different conditions, Different eluent, Eluent, Elution, Elution volume, Elution volumes, Free ligand, Gorner, Hemoglobin, Hemoglobin chromatography, Hemoglobin forms, Hemoglobin solution, Hemoglobin solutions, Hexamethylamine spacer, Human hemoglobin, Hydrated, Immobilised, Immobilised ligand, Ionic interactions, Ligand, Ligand concentrations, Linear regression, Major peak, Marcel dekker, Methb, Molecular mass, Nacl solution, Nitrogen atmosphere, Oxyhb, Oxyhemoglobin, Pharmaceutical faculty, Polyglucose chains, Protein surface, Pyridoxylated hemoglobin, Respiratory mechanism, Saunders company, Sepharose, Spacer, Steric hindrance, Support capacity, Surface charge, Surface charges.
- Teeft :
- Active immobilised, Allosteric, Allosteric site, Amino acids, Association constants, Benzenehexacarboxylic acid, Benzenetetracarboxylic acid, Binding constants, Central cavity, Chromatogr, Chromatographic support, Clinical aspects, Column ligands, Deoxyhb, Deoxyhemoglobin, Deoxyhemoglobin form, Different conditions, Different eluent, Eluent, Elution, Elution volume, Elution volumes, Free ligand, Gorner, Hemoglobin, Hemoglobin chromatography, Hemoglobin forms, Hemoglobin solution, Hemoglobin solutions, Hexamethylamine spacer, Human hemoglobin, Hydrated, Immobilised, Immobilised ligand, Ionic interactions, Ligand, Ligand concentrations, Linear regression, Major peak, Marcel dekker, Methb, Molecular mass, Nacl solution, Nitrogen atmosphere, Oxyhb, Oxyhemoglobin, Pharmaceutical faculty, Polyglucose chains, Protein surface, Pyridoxylated hemoglobin, Respiratory mechanism, Saunders company, Sepharose, Spacer, Steric hindrance, Support capacity, Surface charge, Surface charges.
Abstract
Abstract: A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the pI of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants KPX and KPL (lmol−1) with fixed or free ligand respectively were found: for oxyhemoglobin, KPX=8.0·102, KPL=1.4·104; for deoxyhemoglobin, KPX=9.7·104, KPL=2.3·105. The BHC support capacity was about 4.7·10−5 mol hemoglobing−1 of dry gel corresponding to 1.5·10−6 mol hemoglobing−1 of hydrated gel or 0.1 g hemoglobing−1 of hydrated gel.
Url:
DOI: 10.1016/S0378-4347(97)00113-8
Links to Exploration step
ISTEX:704BD4D68822A5789684C52B6A0D07F6BF4D0597Le document en format XML
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Léonard</name><affiliation><mods:affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Dellacherie, E" sort="Dellacherie, E" uniqKey="Dellacherie E" first="E." last="Dellacherie">E. 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Görner</name><affiliation><mods:affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Gissinger, P" sort="Gissinger, P" uniqKey="Gissinger P" first="P." last="Gissinger">P. Gissinger</name><affiliation><mods:affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Leonard, M" sort="Leonard, M" uniqKey="Leonard M" first="M." last="Léonard">M. Léonard</name><affiliation><mods:affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Dellacherie, E" sort="Dellacherie, E" uniqKey="Dellacherie E" first="E." last="Dellacherie">E. Dellacherie</name><affiliation><mods:affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Chromatography B: Biomedical Sciences and Applications</title><title level="j" type="abbrev">CHROLD</title><idno type="ISSN">0378-4347</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997">1997</date><biblScope unit="volume">694</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="39">39</biblScope><biblScope unit="page" to="48">48</biblScope></imprint><idno type="ISSN">0378-4347</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0378-4347</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Active immobilised</term><term>Allosteric</term><term>Allosteric site</term><term>Amino acids</term><term>Association constants</term><term>Benzenehexacarboxylic acid</term><term>Benzenetetracarboxylic acid</term><term>Binding constants</term><term>Central cavity</term><term>Chromatogr</term><term>Chromatographic support</term><term>Clinical aspects</term><term>Column ligands</term><term>Deoxyhb</term><term>Deoxyhemoglobin</term><term>Deoxyhemoglobin form</term><term>Different conditions</term><term>Different eluent</term><term>Eluent</term><term>Elution</term><term>Elution volume</term><term>Elution volumes</term><term>Free ligand</term><term>Gorner</term><term>Hemoglobin</term><term>Hemoglobin chromatography</term><term>Hemoglobin forms</term><term>Hemoglobin solution</term><term>Hemoglobin solutions</term><term>Hexamethylamine spacer</term><term>Human hemoglobin</term><term>Hydrated</term><term>Immobilised</term><term>Immobilised ligand</term><term>Ionic interactions</term><term>Ligand</term><term>Ligand concentrations</term><term>Linear regression</term><term>Major peak</term><term>Marcel dekker</term><term>Methb</term><term>Molecular mass</term><term>Nacl solution</term><term>Nitrogen atmosphere</term><term>Oxyhb</term><term>Oxyhemoglobin</term><term>Pharmaceutical faculty</term><term>Polyglucose chains</term><term>Protein surface</term><term>Pyridoxylated hemoglobin</term><term>Respiratory mechanism</term><term>Saunders company</term><term>Sepharose</term><term>Spacer</term><term>Steric hindrance</term><term>Support capacity</term><term>Surface charge</term><term>Surface charges</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Active immobilised</term><term>Allosteric</term><term>Allosteric site</term><term>Amino acids</term><term>Association constants</term><term>Benzenehexacarboxylic acid</term><term>Benzenetetracarboxylic acid</term><term>Binding constants</term><term>Central cavity</term><term>Chromatogr</term><term>Chromatographic support</term><term>Clinical aspects</term><term>Column ligands</term><term>Deoxyhb</term><term>Deoxyhemoglobin</term><term>Deoxyhemoglobin form</term><term>Different conditions</term><term>Different eluent</term><term>Eluent</term><term>Elution</term><term>Elution volume</term><term>Elution volumes</term><term>Free ligand</term><term>Gorner</term><term>Hemoglobin</term><term>Hemoglobin chromatography</term><term>Hemoglobin forms</term><term>Hemoglobin solution</term><term>Hemoglobin solutions</term><term>Hexamethylamine spacer</term><term>Human hemoglobin</term><term>Hydrated</term><term>Immobilised</term><term>Immobilised ligand</term><term>Ionic interactions</term><term>Ligand</term><term>Ligand concentrations</term><term>Linear regression</term><term>Major peak</term><term>Marcel dekker</term><term>Methb</term><term>Molecular mass</term><term>Nacl solution</term><term>Nitrogen atmosphere</term><term>Oxyhb</term><term>Oxyhemoglobin</term><term>Pharmaceutical faculty</term><term>Polyglucose chains</term><term>Protein surface</term><term>Pyridoxylated hemoglobin</term><term>Respiratory mechanism</term><term>Saunders company</term><term>Sepharose</term><term>Spacer</term><term>Steric hindrance</term><term>Support capacity</term><term>Surface charge</term><term>Surface charges</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the pI of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants KPX and KPL (lmol−1) with fixed or free ligand respectively were found: for oxyhemoglobin, KPX=8.0·102, KPL=1.4·104; for deoxyhemoglobin, KPX=9.7·104, KPL=2.3·105. The BHC support capacity was about 4.7·10−5 mol hemoglobing−1 of dry gel corresponding to 1.5·10−6 mol hemoglobing−1 of hydrated gel or 0.1 g hemoglobing−1 of hydrated gel.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>ligand</json:string><json:string>allosteric</json:string><json:string>oxyhb</json:string><json:string>deoxyhb</json:string><json:string>allosteric site</json:string><json:string>eluent</json:string><json:string>methb</json:string><json:string>chromatogr</json:string><json:string>deoxyhemoglobin</json:string><json:string>hemoglobin</json:string><json:string>oxyhemoglobin</json:string><json:string>gorner</json:string><json:string>immobilised</json:string><json:string>sepharose</json:string><json:string>elution volume</json:string><json:string>association constants</json:string><json:string>major peak</json:string><json:string>hexamethylamine spacer</json:string><json:string>hemoglobin solutions</json:string><json:string>free ligand</json:string><json:string>column ligands</json:string><json:string>hydrated</json:string><json:string>spacer</json:string><json:string>binding constants</json:string><json:string>chromatographic support</json:string><json:string>surface charges</json:string><json:string>human hemoglobin</json:string><json:string>benzenehexacarboxylic acid</json:string><json:string>immobilised ligand</json:string><json:string>ionic interactions</json:string><json:string>elution</json:string><json:string>different eluent</json:string><json:string>molecular mass</json:string><json:string>central cavity</json:string><json:string>hemoglobin forms</json:string><json:string>steric hindrance</json:string><json:string>different conditions</json:string><json:string>nacl solution</json:string><json:string>amino acids</json:string><json:string>protein surface</json:string><json:string>hemoglobin chromatography</json:string><json:string>nitrogen atmosphere</json:string><json:string>deoxyhemoglobin form</json:string><json:string>surface charge</json:string><json:string>polyglucose chains</json:string><json:string>pharmaceutical faculty</json:string><json:string>benzenetetracarboxylic acid</json:string><json:string>support capacity</json:string><json:string>elution volumes</json:string><json:string>hemoglobin solution</json:string><json:string>ligand concentrations</json:string><json:string>linear regression</json:string><json:string>respiratory mechanism</json:string><json:string>active immobilised</json:string><json:string>pyridoxylated hemoglobin</json:string><json:string>marcel dekker</json:string><json:string>clinical aspects</json:string><json:string>saunders company</json:string></teeft></keywords><author><json:item><name>T. Görner</name><affiliations><json:string>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</json:string></affiliations></json:item><json:item><name>P. Gissinger</name><affiliations><json:string>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</json:string></affiliations></json:item><json:item><name>M. Léonard</name><affiliations><json:string>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</json:string></affiliations></json:item><json:item><name>E. Dellacherie</name><affiliations><json:string>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Hemoglobin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Benzenehexacarboxylic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Benzenetetracarboxylic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Oxyhemoglobin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Deoxyhemoglobin</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the pI of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants KPX and KPL (lmol−1) with fixed or free ligand respectively were found: for oxyhemoglobin, KPX=8.0·102, KPL=1.4·104; for deoxyhemoglobin, KPX=9.7·104, KPL=2.3·105. The BHC support capacity was about 4.7·10−5 mol hemoglobing−1 of dry gel corresponding to 1.5·10−6 mol hemoglobing−1 of hydrated gel or 0.1 g hemoglobing−1 of hydrated gel.</abstract><qualityIndicators><score>7.076</score><pdfVersion>1.1</pdfVersion><pdfPageSize>543 x 743 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>5</keywordCount><abstractCharCount>1220</abstractCharCount><pdfWordCount>5195</pdfWordCount><pdfCharCount>30178</pdfCharCount><pdfPageCount>10</pdfPageCount><abstractWordCount>173</abstractWordCount></qualityIndicators><title>New support for the affinity chromatography of hemoglobin</title><pii><json:string>S0378-4347(97)00113-8</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Chromatography B: Biomedical Sciences and Applications</title><language><json:string>unknown</json:string></language><publicationDate>1997</publicationDate><issn><json:string>0378-4347</json:string></issn><pii><json:string>S0378-4347(00)X0058-8</json:string></pii><volume>694</volume><issue>1</issue><pages><first>39</first><last>48</last></pages><genre><json:string>journal</json:string></genre></host><categories><inist><json:string>sciences appliquees, technologies et medecines</json:string><json:string>sciences biologiques et medicales</json:string><json:string>sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>agronomie. sciences du sol et productions vegetales</json:string></inist></categories><publicationDate>1997</publicationDate><copyrightDate>1997</copyrightDate><doi><json:string>10.1016/S0378-4347(97)00113-8</json:string></doi><id>704BD4D68822A5789684C52B6A0D07F6BF4D0597</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/704BD4D68822A5789684C52B6A0D07F6BF4D0597/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/704BD4D68822A5789684C52B6A0D07F6BF4D0597/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/704BD4D68822A5789684C52B6A0D07F6BF4D0597/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">New support for the affinity chromatography of hemoglobin</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1997 Elsevier Science B.V.</p></availability><date>1997</date></publicationStmt><notesStmt><note type="content">Fig. 1: Chromatograms of hemoglobin (oxyhemoglobin) on a BHC support at different eluent pH. Flow-rate: 22 mlh−1. Column contained 5.5 g of hydrated gel corresponding to 8·10−6 mol of active BHC ligands. Detection at 415 nm.</note><note type="content">Fig. 2: Chromatograms of pyridoxylhemoglobin on BHC support at different eluent pH. Flow-rate: 22 mlh−1. Column contained 5.5 g of hydrated gel corresponding to 8·10−6 mol of active BHC ligands. Detection at 415 nm.</note><note type="content">Fig. 3: Determination of oxyhemoglobin affinity constants at pH 6.9 with free and fixed BHC ligands, KPL and KPX, respectively. Quantity of active immobilised BHC ligands on the column Qx=1.4·10−5 mol. KPL=1.4·104 lmol−1; KPX=8.0·102 lmol−1.</note><note type="content">Fig. 4: Determination of deoxyhemoglobin affinity constants at pH 6.9 with free and fixed BHC ligands, KPL and KPX, respectively: Quantity of active immobilised BHC ligands on the column Qx=1.4·10−5 mol. KPL=2.3·105 lmol−1; KPX=9.7·104 lmol−1.</note><note type="content">Table 1: Hemoglobin association constants with fixed or free benzenehexacarboxylic ligands, KPX and KPL respectively, at pH 6.9</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">New support for the affinity chromatography of hemoglobin</title><author xml:id="author-0000"><persName><forename type="first">T.</forename><surname>Görner</surname></persName><note type="correspondence"><p>Corresponding author.</p></note><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation></author><author xml:id="author-0001"><persName><forename type="first">P.</forename><surname>Gissinger</surname></persName><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation></author><author xml:id="author-0002"><persName><forename type="first">M.</forename><surname>Léonard</surname></persName><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation></author><author xml:id="author-0003"><persName><forename type="first">E.</forename><surname>Dellacherie</surname></persName><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation></author><idno type="istex">704BD4D68822A5789684C52B6A0D07F6BF4D0597</idno><idno type="DOI">10.1016/S0378-4347(97)00113-8</idno><idno type="PII">S0378-4347(97)00113-8</idno></analytic><monogr><title level="j">Journal of Chromatography B: Biomedical Sciences and Applications</title><title level="j" type="abbrev">CHROLD</title><idno type="pISSN">0378-4347</idno><idno type="PII">S0378-4347(00)X0058-8</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997"></date><biblScope unit="volume">694</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="39">39</biblScope><biblScope unit="page" to="48">48</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1997</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the pI of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants KPX and KPL (lmol−1) with fixed or free ligand respectively were found: for oxyhemoglobin, KPX=8.0·102, KPL=1.4·104; for deoxyhemoglobin, KPX=9.7·104, KPL=2.3·105. The BHC support capacity was about 4.7·10−5 mol hemoglobing−1 of dry gel corresponding to 1.5·10−6 mol hemoglobing−1 of hydrated gel or 0.1 g hemoglobing−1 of hydrated gel.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Hemoglobin</term></item><item><term>Benzenehexacarboxylic acid</term></item><item><term>Benzenetetracarboxylic acid</term></item><item><term>Oxyhemoglobin</term></item><item><term>Deoxyhemoglobin</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1997-01-29">Modified</change><change when="1997">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/704BD4D68822A5789684C52B6A0D07F6BF4D0597/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>CHROLD</jid><aid>9065</aid><ce:pii>S0378-4347(97)00113-8</ce:pii><ce:doi>10.1016/S0378-4347(97)00113-8</ce:doi><ce:copyright year="1997" type="full-transfer">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>New support for the affinity chromatography of hemoglobin</ce:title><ce:author-group><ce:author><ce:given-name>T.</ce:given-name><ce:surname>Görner</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>P.</ce:given-name><ce:surname>Gissinger</ce:surname></ce:author><ce:author><ce:given-name>M.</ce:given-name><ce:surname>Léonard</ce:surname></ce:author><ce:author><ce:given-name>E.</ce:given-name><ce:surname>Dellacherie</ce:surname></ce:author><ce:affiliation><ce:textfn>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author.</ce:text></ce:correspondence></ce:author-group><ce:date-received day="12" month="6" year="1996"></ce:date-received><ce:date-revised day="29" month="1" year="1997"></ce:date-revised><ce:date-accepted day="6" month="2" year="1997"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the p<ce:italic>I</ce:italic> of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants <ce:italic>K</ce:italic><ce:inf>PX</ce:inf> and <ce:italic>K</ce:italic><ce:inf>PL</ce:inf> (l<ce:hsp sp="0.25"></ce:hsp>mol<ce:sup>−1</ce:sup>) with fixed or free ligand respectively were found: for oxyhemoglobin, <ce:italic>K</ce:italic><ce:inf>PX</ce:inf>=8.0·10<ce:sup>2,</ce:sup> <ce:italic>K</ce:italic><ce:inf>PL</ce:inf>=1.4·10<ce:sup>4</ce:sup>; for deoxyhemoglobin, <ce:italic>K</ce:italic><ce:inf>PX</ce:inf>=9.7·10<ce:sup>4</ce:sup>, <ce:italic>K</ce:italic><ce:inf>PL</ce:inf>=2.3·10<ce:sup>5</ce:sup>. The BHC support capacity was about 4.7·10<ce:sup>−5</ce:sup> mol hemoglobin<ce:hsp sp="0.25"></ce:hsp>g<ce:sup>−1</ce:sup> of dry gel corresponding to 1.5·10<ce:sup>−6</ce:sup> mol hemoglobin<ce:hsp sp="0.25"></ce:hsp>g<ce:sup>−1</ce:sup> of hydrated gel or 0.1 g hemoglobin<ce:hsp sp="0.25"></ce:hsp>g<ce:sup>−1</ce:sup> of hydrated gel.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Hemoglobin</ce:text></ce:keyword><ce:keyword><ce:text>Benzenehexacarboxylic acid</ce:text></ce:keyword><ce:keyword><ce:text>Benzenetetracarboxylic acid</ce:text></ce:keyword><ce:keyword><ce:text>Oxyhemoglobin</ce:text></ce:keyword><ce:keyword><ce:text>Deoxyhemoglobin</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>New support for the affinity chromatography of hemoglobin</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>New support for the affinity chromatography of hemoglobin</title></titleInfo><name type="personal"><namePart type="given">T.</namePart><namePart type="family">Görner</namePart><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation><description>Corresponding author.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">P.</namePart><namePart type="family">Gissinger</namePart><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">M.</namePart><namePart type="family">Léonard</namePart><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">E.</namePart><namePart type="family">Dellacherie</namePart><affiliation>Laboratoire de Chimie-Physique Macromoléculaire, CNRS URA 494/ENSIC, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1997</dateIssued><dateModified encoding="w3cdtf">1997-01-29</dateModified><copyrightDate encoding="w3cdtf">1997</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently bound to the spacer arm. At pH close to the pI of the protein, the biospecificity of the support due to the interactions of the allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand. The following values of binding constants KPX and KPL (lmol−1) with fixed or free ligand respectively were found: for oxyhemoglobin, KPX=8.0·102, KPL=1.4·104; for deoxyhemoglobin, KPX=9.7·104, KPL=2.3·105. The BHC support capacity was about 4.7·10−5 mol hemoglobing−1 of dry gel corresponding to 1.5·10−6 mol hemoglobing−1 of hydrated gel or 0.1 g hemoglobing−1 of hydrated gel.</abstract><note type="content">Fig. 1: Chromatograms of hemoglobin (oxyhemoglobin) on a BHC support at different eluent pH. Flow-rate: 22 mlh−1. Column contained 5.5 g of hydrated gel corresponding to 8·10−6 mol of active BHC ligands. Detection at 415 nm.</note><note type="content">Fig. 2: Chromatograms of pyridoxylhemoglobin on BHC support at different eluent pH. Flow-rate: 22 mlh−1. Column contained 5.5 g of hydrated gel corresponding to 8·10−6 mol of active BHC ligands. Detection at 415 nm.</note><note type="content">Fig. 3: Determination of oxyhemoglobin affinity constants at pH 6.9 with free and fixed BHC ligands, KPL and KPX, respectively. Quantity of active immobilised BHC ligands on the column Qx=1.4·10−5 mol. KPL=1.4·104 lmol−1; KPX=8.0·102 lmol−1.</note><note type="content">Fig. 4: Determination of deoxyhemoglobin affinity constants at pH 6.9 with free and fixed BHC ligands, KPL and KPX, respectively: Quantity of active immobilised BHC ligands on the column Qx=1.4·10−5 mol. KPL=2.3·105 lmol−1; KPX=9.7·104 lmol−1.</note><note type="content">Table 1: Hemoglobin association constants with fixed or free benzenehexacarboxylic ligands, KPX and KPL respectively, at pH 6.9</note><subject><genre>Keywords</genre><topic>Hemoglobin</topic><topic>Benzenehexacarboxylic acid</topic><topic>Benzenetetracarboxylic acid</topic><topic>Oxyhemoglobin</topic><topic>Deoxyhemoglobin</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Chromatography B: Biomedical Sciences and Applications</title></titleInfo><titleInfo type="abbreviated"><title>CHROLD</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">19970620</dateIssued></originInfo><identifier type="ISSN">0378-4347</identifier><identifier type="PII">S0378-4347(00)X0058-8</identifier><part><date>19970620</date><detail type="volume"><number>694</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>252</end></extent><extent unit="pages"><start>39</start><end>48</end></extent></part></relatedItem><identifier type="istex">704BD4D68822A5789684C52B6A0D07F6BF4D0597</identifier><identifier type="ark">ark:/67375/6H6-ZZ3R5X96-M</identifier><identifier type="DOI">10.1016/S0378-4347(97)00113-8</identifier><identifier type="PII">S0378-4347(97)00113-8</identifier><accessCondition type="use and reproduction" contentType="copyright">©1997 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©1997</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/document/704BD4D68822A5789684C52B6A0D07F6BF4D0597/metadata/json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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