Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.
Anal Biochem
; 511: 42-51, 2016 10 15.
Article
em En
| MEDLINE
| ID: mdl-27485269
ABSTRACT
This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Sulfonamidas
/
Biotina
/
Anidrases Carbônicas
/
Enzimas Imobilizadas
Limite:
Humans
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article