Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Huang, Zhijian

doi:10.1021/bi00099a005

Cited by 25 publications

(17 citation statements)

References 9 publications

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“…At pH7. [20,211, but E~~~ of Huang [19] is higher by a factor of about 2.7. In our calculations of concentrations, the mean values of E~~~ = 66.74 mM-'cm-' at pH 7.0 and E~~~ = 73.96 mM-'cm-' at pH 7.5 were used.…”

Section: Results and Discussion Molar Absorbance Of Fluoresceinmentioning

confidence: 91%

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No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

Fiedler¹,

Hinz²

1994

European Journal of Biochemistry

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In the enzymic hydrolysis of biopolymers such as polypeptides, polysaccharides or nucleic acids, two extreme patterns of action are conceivable [l]. The enzyme, after forming an enzyme-substrate complex, may begin to catalyze hydrolysis of mult.iple bonds in what has been described as zipper fashion until it comes to the end of the chain. Alternatively, the hydrolysis of only one bond per effective encounter is catalyzed, the classical random action. Between these extremes is the case of 'multiple attack', where several bonds are hydrolyzed before the enzyme-substrate complex dissociates. Examples for the hydrolysis of more than one bond per enzyme-substrate encounter seem to be relatively rare. Multiple attack has been reported in the hydrolysis of starch by a-and P-amylases [l, 21. 'Processivity' (as it is called there) is observed in enzymes processing the highly charged DNAs or RNAs and might be a feature of their polymerase function, retained e.g. in the partially processive hydrolysis by the RNase H activity of retroviral reverse transcriptase [3]. Processive hydrolysis is also found with ribonuclease I1 [4] and exonuclease I from Escherichiu coli and some further exonucleases As to cases where only a few specific bonds are to be hydrolyzed in a substrate molecule, partial processive cleavage was described in the hydrolysis of polynucleotides with two EcoRI sites by EcoRI endonuclease [6, 71. We have obtained evidence that in the release of the kallidin decapeptide from bovine kininogen by tissue kallikrein the two peptide bonds hydrolyzed may be cleaved at a single enzyme-substrate encounter [8, 91. Therefore, we were highly interested in a recent report [lo] stating that in the hydrolysis of fluorescein di-P-galactoside (FDG) by P-galactosidase intermediate channelling does occur.

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Section: Results and Discussion Molar Absorbance Of Fluoresceinmentioning

confidence: 91%

“…The molar absorbance of fluorescein is therefore crucial. Huang [19] determined a value of E~~~ = 198 mM-'cm-' at pH 7.5, while another value from the literature is E~~~ = 50.3 ? 2.0 mM-'cm-' at pH 7 [20,211.…”

Section: Results and Discussion Molar Absorbance Of Fluoresceinmentioning

confidence: 99%

No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

Fiedler¹,

Hinz²

1994

European Journal of Biochemistry

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“…FMG is one common fluorogenic substrate for β-Gal. However, FMG has strong fluorescence and absorption at the wavelength where its enzymatic breakdown product, fluorescein, is measured [25,26]. Therefore, measurement of fluorescein production in the presence of FMG is difficult with a traditional microtiter plate assay.…”

Section: Hongwei Xu · Andrew G Ewingmentioning

confidence: 99%

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Ewing

2004

Analytical and Bioanalytical Chemistry

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The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono- beta- d-galactopyranoside (FMG) by beta- d-galactosidase ( beta-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about beta-Gal has been successfully obtained by varying the concentration of FMG. beta-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of beta-Gal by phenylethyl beta- d-thiogalactoside (PETG) and beta-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.

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“…The intermediate, fluorescein mono-b-D-galactopyranoside (FMG) 10, although a good substrate for bGal, has a strong fluorescence and absorbance at the wavelength used for detection of fluorescein thereby hampering its utility. 74 Nolan et al demonstrated that 9 could distinguish between lacZ+ and lacZÀ cells using fluorescence-activated cell sorter (FACS) analysis. 72 MacDonald reported use of 9 in a multiwell plate system, demonstrating the higher sensitivity of 9 compared with colourimetric o-NPG and fluorogenic 4-MU, although it was noted that the turnover rate is slower due to the requirement for sequential glycosidic bond hydrolysis.…”

Section: Fluoresceinmentioning

confidence: 99%

Recent advances in the development of synthetic chemical probes for glycosidase enzymes

2015

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The emergence of synthetic glycoconjugates as chemical probes for the detection of glycosidase enzymes has resulted in the development of a range of useful chemical tools with applications in glycobiology, biotechnology, medical and industrial research. Critical to the function of these probes is the preparation of substrates containing a glycosidic linkage that when activated by a specific enzyme or group of enzymes, irreversibly releases a reporter molecule that can be detected. Starting from the earliest examples of colourimetric probes, increasingly sensitive and sophisticated substrates have been reported. In this review we present an overview of the recent advances in this field, covering an array of strategies including chromogenic and fluorogenic substrates, lanthanide complexes, gels and nanoparticles. The applications of these substrates for the detection of various glycosidases and the scope and limitations for each approach are discussed.

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Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Cited by 25 publications

References 9 publications

No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Recent advances in the development of synthetic chemical probes for glycosidase enzymes

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