| dc.contributor.author |
Mazitsos, CF |
en |
| dc.contributor.author |
Rigden, DJ |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:45:57Z |
|
| dc.date.available |
2014-06-06T06:45:57Z |
|
| dc.date.issued |
2004 |
en |
| dc.identifier.issn |
00219673 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1016/j.chroma.2003.09.048 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/2723 |
|
| dc.subject |
Affinity adsorbents |
en |
| dc.subject |
Biomimetic ligands |
en |
| dc.subject |
Dyes |
en |
| dc.subject |
Enzymes |
en |
| dc.subject |
Galactose dehydrogenase |
en |
| dc.subject |
Molecular modeling |
en |
| dc.subject |
Pseudomonas fluorescens |
en |
| dc.subject |
Triazines |
en |
| dc.subject.other |
Adsorbents |
en |
| dc.subject.other |
Chemical bonds |
en |
| dc.subject.other |
Chromatographic analysis |
en |
| dc.subject.other |
Electrophoresis |
en |
| dc.subject.other |
Hydrophilicity |
en |
| dc.subject.other |
Muscle |
en |
| dc.subject.other |
Yeast |
en |
| dc.subject.other |
Ligands |
en |
| dc.subject.other |
Enzymes |
en |
| dc.subject.other |
1,1 carbonyldiimidazole |
en |
| dc.subject.other |
adsorbent |
en |
| dc.subject.other |
agarose |
en |
| dc.subject.other |
aliphatic compound |
en |
| dc.subject.other |
anthraquinone |
en |
| dc.subject.other |
beta galactose dehydrogenase |
en |
| dc.subject.other |
dodecyl sulfate sodium |
en |
| dc.subject.other |
galactosamine |
en |
| dc.subject.other |
galactose dehydrogenase |
en |
| dc.subject.other |
imidazole derivative |
en |
| dc.subject.other |
nicotinamide adenine dinucleotide |
en |
| dc.subject.other |
shikimic acid |
en |
| dc.subject.other |
triazine |
en |
| dc.subject.other |
unclassified drug |
en |
| dc.subject.other |
affinity chromatography |
en |
| dc.subject.other |
amino terminal sequence |
en |
| dc.subject.other |
animal cell |
en |
| dc.subject.other |
animal tissue |
en |
| dc.subject.other |
anion exchange chromatography |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
chimera |
en |
| dc.subject.other |
chromatography |
en |
| dc.subject.other |
dissociation constant |
en |
| dc.subject.other |
enzyme binding |
en |
| dc.subject.other |
enzyme purification |
en |
| dc.subject.other |
hydrophilicity |
en |
| dc.subject.other |
muscle |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
pea |
en |
| dc.subject.other |
polyacrylamide gel electrophoresis |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
Pseudomonas fluorescens |
en |
| dc.subject.other |
rabbit |
en |
| dc.subject.other |
Saccharomyces cerevisiae |
en |
| dc.subject.other |
Chromatography, Affinity |
en |
| dc.subject.other |
Chromatography, Ion Exchange |
en |
| dc.subject.other |
Coloring Agents |
en |
| dc.subject.other |
Electrophoresis, Polyacrylamide Gel |
en |
| dc.subject.other |
Galactose Dehydrogenases |
en |
| dc.subject.other |
Ligands |
en |
| dc.subject.other |
Pseudomonas fluorescens |
en |
| dc.subject.other |
Oryctolagus cuniculus |
en |
| dc.subject.other |
Pisum sativum |
en |
| dc.subject.other |
Pseudomonas |
en |
| dc.subject.other |
Pseudomonas fluorescens |
en |
| dc.title |
Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens β-galactose dehydrogenase |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1016/j.chroma.2003.09.048 |
en |
| heal.publicationDate |
2004 |
en |
| heal.abstract |
Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH). The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer'. The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents. Both immobilized mimodyes successfully bound P. fluorescens GaDH but failed to bind the enzyme from rabbit muscle. Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so. The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P. fluorescens GaDH. The dissociation constants (KD) of BM1 and BM2 for P. fluorescens GaDH were determined by analytical affinity chromatography to be 5.9μM and 15.4μM, respectively. The binding capacities of adsorbents BM1 and BM2 were 18U/mg adsorbent and 6U/mg adsorbent, respectively. Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P. fluorescens GaDH. Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively. The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854U/mg, respectively. The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. © 2004 Elsevier B.V. All rights reserved. |
en |
| heal.journalName |
Journal of Chromatography A |
en |
| dc.identifier.issue |
1-2 |
en |
| dc.identifier.volume |
1029 |
en |
| dc.identifier.doi |
10.1016/j.chroma.2003.09.048 |
en |
| dc.identifier.spage |
103 |
en |
| dc.identifier.epage |
112 |
en |