Peer-Reviewed Journal Details
Mandatory Fields
Briciu-Burghina, C;Heery, B;Regan, F
2015
January
The Analyst
Continuous fluorometric method for measuring beta-glucuronidase activity: comparative analysis of three fluorogenic substrates
Published
10 ()
Optional Fields
EXCITED-STATE REACTIONS ESCHERICHIA-COLI ENZYME FLUORESCENCE COUMARINS ACID GUS 7-HYDROXY-4-METHYLCOUMARIN 7-HYDROXYCOUMARINS IDENTIFICATION
140
5953
5964
E. coli beta-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl- umbelliferyl-beta-D-glucuronide (6-CMUG) were determined experimentally (K-m = 0.11 mM, K-cat = 74 s(-1), K-cat/K-m = 6.93 x 10(5) s(-1) M-1) and compared with the ones found for 4-methyl-umbelliferyl-beta-D-glucuronide (4-MUG) (K-m = 0.07 mM, K-cat = 92 s(-1), K-cat/K-m = 1.2(9) x 10(6) s(-1) M-1) and 3-carboxy-umbelliferyl-beta-D-glucuronide (3-CUG) (K-m = 0.48 mM, K-cat = 35 s(-1), K-cat/K-m = 7.40 x 10(4) s(-1) M-1). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.
CAMBRIDGE
0003-2654
10.1039/c5an01021g
Grant Details