Measurement of hydrolysis kinetics of galactose-substituted fluorescein by β-galactosidase at the toluene—water interface by spinning microtube fluorometry

Okamoto, Yusuke; Monjushiro, Hideaki; Fukumoto, Takao; Watarai, Hitoshi
September 2006
Analytical & Bioanalytical Chemistry;Sep2006, Vol. 385 Issue 8, p1430
Academic Journal
A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis of 5-dodecanoylaminofluorescein di-β- D-galactopyranoside (C12FDG) by β-galactosidase (β-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 1.4×102 and 1.97, respectively. The interfacial adsorption constants from the toluene phase to the interface at pH 7.3 were 4.8×10−4 and 1.7×10−2 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 β-gal and that the hydrolysis reaction rate constant at the interface at pH 7.3 was 1.84×103 M−1s−1, almost equal to that in the aqueous solution, 1.76×103 M−1s−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%.


Related Articles

  • High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae ( Bombyx mori). Jie Zhu; Yuguan Ze; Chi Zhang; Yuhui Zang; Haiqin Lu; Ping Chu; Manji Sun; Junchuan Qin // Applied Microbiology & Biotechnology;Aug2006, Vol. 72 Issue 1, p103 

    Human serum paraoxonase 1 (hPON1) belongs to a family of enzymes that catalyze the hydrolysis of a broad range of esters and lactones. Although the very first identification of hPON1 might have been as a calcium-dependent paraoxonase/arylesterase, PON1 is in fact a lactonase associated with...

  • Back to (non)-Basics: Recent Developments in Neutral and Charge- Balanced Glycosidase Inhibitors. Houston, Todd A.; Blanchfield, Joanne T. // Mini Reviews in Medicinal Chemistry;Nov2003, Vol. 3 Issue 7, p669 

    Certain glycosidase inhibitors possess potent antiviral, antitumour and antidiabetic properties. Glyconic acid lactones, the earliest glycosidase inhibitors identified, have planar anomeric carbons that mimic the transition state of glycoside hydrolysis. Other classes include lactams, glycals,...

  • Fluorometric assay of lipase by some new synthetized fluorescein aralkyl esters. Eshghi, Hossein; Ghafoorian, Fatemeh; Bakavoli, Mahdi // Research on Chemical Intermediates;Aug2015, Vol. 41 Issue 8, p5439 

    A series of fluorescein aralkyl esters were synthesized through esterification of fluorescein with carboxylic acids or acyl chlorides and applied to the fluorometric assay of lipase. The rate of spontaneous hydrolysis and enzymatic hydrolysis was measured by monitoring of fluorescence...

  • Kinetic gravimetric sorption of low volatility gases and vapors in polymers. Dhoot, Sushil N.; Freeman, Benny D. // Review of Scientific Instruments;Dec2003, Vol. 74 Issue 12, p5173 

    A kinetic gravimetric sorption apparatus for measuring solubility and diffusivity of low vapor pressure gases and vapors in polymers and other sorbents is described. Sorption and transport data for toluene vapor in poly(dimethyl siloxane) at 25 °C are provided to illustrate the method. These...

  • Removal of Parabens from Aqueous Solution Using β-Cyclodextrin Cross-Linked Polymer. Yuk Ping Chin; Mohamad, Sharifah; Abas, Mhd. Radzi Bin // International Journal of Molecular Sciences;Sep2010, Vol. 11 Issue 9, p3459 

    The removal of four parabens, methyl-, ethyl-, propyl-, and benzyl-paraben, by β-cyclodextrin (β-CD) polymer from aqueous solution was studied. Different β-CD polymers were prepared by using two cross-linkers, i.e., hexamethylene diisocyanate (HMDI) and toluene-2,6-diisocyanate (TDI),...

  • Adsorption of a cationic porphyrin onto mesoporous silicas. Yoshida, Atsuko; Kakegawa, Norishige; Ogawa, Makoto // Research on Chemical Intermediates;2003, Vol. 29 Issue 7-9, p721 

    The adsorption of a cationic porphyrin, tetrakis-(N-methyl-4-pyridiniumyl)porphine, into mesoporous silicas from solution of tetrakis-(N-methyl-4-pyridiniumyl)porphine p-toluene sulfonate was investigated. Irrespective of the pore size (2.4, 3.5 and 4.2 nm), the cationic porphyrin was adsorbed...

  • Improvement in the GC–MS method for determining urinary toluene-diamine and its application to the biological monitoring of workers exposed to toluene-diisocyanate. Sakai, Tadashi; Morita, Yoko; Jaehoon Roh; Hyoungryoul Kim; Yangho Kim // International Archives of Occupational & Environmental Health;Sep2005, Vol. 78 Issue 6, p459 

    Objectives: To develop a simple and sensitive GC–MS method for determining toluene-diamine (TDA) in urine and to apply the method for biological monitoring of workers exposed to toluene-diisocyanate (TDI). Methods: After acid hydrolysis of 0.1 ml of urine, diluted tenfold with water, for...

  • Kinetics and mechanism of hydrolysis of 3-diazobenzofuran-2-one and its hydrolysis product (3-hydroxybenzofuran-2-one). Chiang, Y; Kresge, A J; Meng, Q // Canadian Journal of Chemistry;Jan2002, Vol. 80 Issue 1, p82 

    Rates of acid-catalyzed hydrolysis of 3-diazobenzofuran-2-one, measured in concentrated aqueous perchloric acid and hydrochloric acid solutions, were found to correlate well with the Cox–Yates X[sub o] excess acidity function, giving k[sub H+] = 1.66 × 10[sup –4] M[sup...

  • Reduction of Alkyl-2-amino-5,6-dialkyl-3-cyanopyridine-4-carboxylates. Vasil�ev, A.; Lyshchikov, A.; Nasakin, O.; Kayukov, Ya.; Tafeenko, V. // Russian Journal of Organic Chemistry;Feb2005, Vol. 41 Issue 2, p279 

    Reduction of alkyl-2-amino-5,6-dialkyl-3-cyanopyridine-4-carboxylates with sodium borohydride in protic solvents gave rise to 4-amino-3-imino-6,7-dialkyl-1,3-dihydrofuro[3,4- c]pyridines that at hydrolysis in acid medium afforded the corresponding lactones.


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics