Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Carbohydrate Research Année : 2012

Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient.

Résumé

Fluorescein monoglycosides (d-galactopyranoside (FMG) and d-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated β-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K(m)) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K(m) value for biotinylated β-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5×10(-10)m(2)s(-1). Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent.

Domaines

Chimie organique

Dates et versions

hal-00723344 , version 1 (09-08-2012)

Identifiants

Citer

Prasun K Mandal, Laurent Cattiaux, David Bensimon, Jean-Maurice Mallet. Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient.. Carbohydrate Research, 2012, 358, pp.40-46. ⟨10.1016/j.carres.2012.05.017⟩. ⟨hal-00723344⟩
65 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More