Measurement of hydrolysis kinetics of galactose-substituted fluorescein by beta-galactosidase at the toluene-water interface by spinning microtube fluorometry.
A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis of 5-dodecanoylaminofluorescein di-beta-D: -galactopyranoside (C12FDG) by beta-galactosidase (beta-gal) in the toluene-water system. The nonfluorescent lactone form of C12FDG in the toluene phase was converted at the interface to 5-dodecanoylaminofluorescein (C12F), which was fluorescent in the aqueous phase as a dianion at pH 7.3, though some part of C12F was extracted into the toluene phase as its nonfluorescent lactone form. The distribution ratios of C12FDG and C12F at pH 7.3 were determined as 1.4 x 10(2) and 1.97, respectively. The interfacial adsorption constants from the toluene phase to the interface at pH 7.3 were 4.8 x 10(-4) and 1.7 x 10(-2) dm for C12FDG and C12F, respectively. The kinetic experiments with the SMF method concluded that the rate-determining step of the enzymatic hydrolysis at the interface and in the aqueous phase was the 1:1 reaction of C12FDG and beta-gal and that the hydrolysis reaction rate constant at the interface at pH 7.3 was 1.84 x 10(3) M(-1)s(-1), almost equal to that in the aqueous solution, 1.76 x 10(3) M(-1)s(-1). Finally, the SMF method revealed that the contribution of the interfacial reaction to the overall hydrolysis reaction rate of the toluene-water system was as high as 97%.
Okamoto
Yusuke
Y
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
Monjushiro
Hideaki
H
Fukumoto
Takao
T
Watarai
Hitoshi
H
D004795:Enzyme Stability; D019793:Fluorescein / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D005690:Galactose / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008956:Models, Chemical; D014050:Toluene / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism*
D014867:Water; D014050:Toluene; D001616:beta-Galactosidase; D019793:Fluorescein; D005690:Galactose