期刊
CARBOHYDRATE RESEARCH
卷 358, 期 -, 页码 40-46出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.carres.2012.05.017
关键词
Galactoside; Glucoside; Fluorescein; Biotinylated galactosidase; Fluorogenic probe; Translational diffusion coefficient
资金
- Marie Curie International Incoming Fellowship
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 beta-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 beta-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 x 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. (C) 2012 Elsevier Ltd. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据